Methods for inhibiting immune complex formation in a subject

ABSTRACT

Polypeptides and other compounds that can bind specifically to the C H 2-C H 3 cleft of an immunoglobulin molecule, and methods for using such polypeptides and compounds to inhibit Fc-mediated immune complex formation, are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part and claims benefit under 35U.S.C. § 119(a) of International Application No. PCT/US2005/008131,having an International Filing Date of Mar. 10, 2005, which published inEnglish as International Publication Number WO 2005/086947, and whichclaims the benefit of priority of U.S. Provisional Application Ser. No.60/551,817 having a filing date of Mar. 10, 2004.

TECHNICAL FIELD

This invention relates to inhibition of immune complex formation, andmore particularly to inhibition of immune complex formation bypolypeptides and other small molecules.

BACKGROUND

Humoral immune responses are triggered when an antigen bindsspecifically to an antibody. The combination of an antibody molecule andan antigen forms a small, relatively soluble immune complex. Antigenseither can be foreign substances, such as viral or bacterialpolypeptides, or can be “self-antigens” such as polypeptides normallyfound in the human body. The immune system normally distinguishesforeign antigens from self-antigens. “Autoimmune” disease can occur,however, when this system breaks down, such that the immune system turnsupon the body and destroys tissues or organ systems as if they wereforeign substances. Examples of this process include the destructionofjoints in rheumatoid arthritis (RA) and the destruction of the kidneysin lupus nephritis. Larger immune complexes are more pathogenic thansmall, more soluble immune complexes. The formation of large, relativelyinsoluble immune complexes can result from both the interaction ofantibody molecules with antigen and the interaction of antibodymolecules with each other. Such immune complexes also can result frominteractions between antibodies in the absence of antigen.

Antibodies can prevent infections by coating viruses or bacteria, butotherwise are relatively harmless by themselves. In contrast, organspecific tissue damage can occur when antibodies combine with antigensand the resulting immune complexes bind to certain effector molecules inthe body. Effector molecules are so named because they carry out thepathogenic effects of immune complexes. By inhibiting the formation oflarge, insoluble immune complexes, or by inhibiting the binding ofimmune complexes to effector molecules, the tissue damaging effects ofimmune complexes could be prevented.

SUMMARY

This invention is based on the discovery that polypeptides having aminoacid sequences based on that set forth in SEQ ID NO:1 can bindspecifically and with high affinity to the C_(H)2-C_(H)3 domain of animmunoglobulin molecule, thus inhibiting the formation of insolubleimmune complexes containing antibodies and antigens, and preventing thebinding of such complexes to effector molecules. The invention providessuch polypeptides, as well as methods for using the polypeptides andcompounds to inhibit immune complex formation and treat autoimmunedisorders such as rheumatoid arthritis.

In one aspect, the invention features a method for inhibiting immunecomplex formation in a subject. The method can include administering tothe subject a composition containing a purified polypeptide, wherein thepolypeptide includes the amino acid sequenceCys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:8), andwherein Xaa is Arg, Trp, Tyr, or Phe. The immune complex formation canbe associated with rheumatoid arthritis. The method can further includethe step of monitoring the subject for clinical or molecularcharacteristics of rheumatoid arthritis.

The polypeptide can further contain a terminal stabilizing group. Theterminal stabilizing group can be at the amino terminus or the carboxyterminus of the polypeptide, or both, and can be a tripeptide having theamino acid sequence Xaa-Pro-Pro, wherein Xaa is any amino acid (e.g.,Ala). The terminal stabilizing group can be a small stable protein(e.g., a four-helix bundle protein such as Rop). The polypeptide canfurther include an additional amino acid at the amino terminus of theamino acid sequence. The additional amino acid can be any amino acidother than Cys (e.g., the amino terminal amino acid can be Asp).

The polypeptide can have a length of about 10 to about 50 amino acids.The polypeptide can include the amino acid sequenceAsp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:2). Thepolypeptide can include the amino acid sequenceTrp-Glu-Ala-Asp-Cys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Lys-Val-Glu-Glu(SEQ ID NO:32).

The invention also features a method for treating rheumatoid arthritis.The method can include identifying an individual with rheumatoidarthritis or at risk for developing rheumatoid arthritis, andadministering to the individual a composition containing a purifiedpolypeptide containing the amino acid sequenceCys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:8), whereinXaa is Arg, Trp, Tyr, or Phe. The method can further include the step ofmonitoring the subject for clinical or molecular characteristics ofrheumatoid arthritis.

The polypeptide can further include an Asp at the amino terminus of theamino acid sequence. The polypeptide can further include a terminalstabilizing group. The terminal stabilizing group can be at the aminoterminus or the carboxy terminus of the polypeptide, or both, and can bea tripeptide having the amino acid sequence Xaa-Pro-Pro, wherein Xaa isany amino acid (e.g., Ala). The terminal stabilizing group can be asmall stable protein (e.g., a four-helix bundle protein such as Rop).

The polypeptide can have a length of about 10 to about 50 amino acids.The polypeptide can contain the amino acid sequenceAsp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:2). Thepolypeptide can contain the amino acid sequenceTrp-Glu-Ala-Asp-Cys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Lys-Val-Glu-Glu(SEQ ID NO:32).

In another aspect, the invention features a purified polypeptidecontaining the amino acid sequenceXaa₁-Pro-Pro-Cys-Ala-Xaa₂-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ IDNO:12), wherein Xaa₁ is any amino acid (e.g., Ala) and Xaa₂ is Arg, Trp,Tyr, or Phe. The invention also features a composition containing thepolypeptide.

In another aspect, the invention features a purified polypeptidecontaining the amino acid sequenceCys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:9). Thepurified polypeptide can have a length of no more than about 20 aminoacids. The purified polypeptide can further contain a terminalstabilizing group. The terminal stabilizing group can be at the aminoterminus or the carboxy terminus of the polypeptide, or both, and can bea tripeptide with the amino acid sequence Xaa-Pro-Pro, wherein Xaa isany amino acid (e.g., Ala). The terminal stabilizing group can be asmall stable protein (e.g., a four-helix bundle protein such as Rop).The purified polypeptide can further contain an Asp at the aminoterminus of the amino acid sequence. The invention also features acomposition containing the purified polypeptide.

In yet another aspect, the invention features a purified polypeptide,the amino acid sequence of which consists of:(Xaa₁)_(n)-Xaa₂-Cys-Ala-Xaa₃-His-Xaa₄-Xaa₅-Xaa₆-Leu-Val-Trp-Cys-(Xaa₇)_(n)(SEQ ID NO:1), wherein Xaa₁ is absent or is any amino acid, Xaa₂ is Pheor Arg, Xaa₃ is any amino acid, Xaa₄ is Gly or Ala, Xaa₅ is Glu or Ala,and Xaa₆ is any non-aromatic amino acid.

In another aspect, the invention features a purified polypeptide, theamino acid sequence of which consists of:(Xaa₁)_(n)-Cys-Ala-Xaa₂-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-(Xaa₃)_(n)(SEQ ID NO:35), wherein Xaa₁ is any amino acid, Xaa₂ is Arg, Trp, Tyr,or Phe, Xaa₃ is any amino acid, and n is 0, 1, 2, 3, 4, or 5.

In still another aspect, the invention features a purified polypeptide,the amino acid sequence of which consists of:(Xaa₁)_(n)-Cys-Ala-Xaa₂-His-Xaa₃-Xaa₄-Xaa₅-Leu-Val-Trp-Cys-Xaa₆-(Xaa₇)_(n)(SEQ ID NO:34), wherein Xaa₁ is any amino acid, Xaa₂ is Phe or Arg, Xaa₃is any amino acid, Xaa₄ is Gly or Ala, Xaa₅ is Glu or Ala, Xaa₆ is anynon-aromatic amino acid, Xaa₇ is any amino acid, and n is 0, 1, 2, 3, 4,or 5.

In another aspect, the invention features a purified polypeptide, theamino acid sequence of which consists of:(Xaa₁)_(n)-Cys-Ala-Xaa₂-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-(Xaa₃)_(n)(SEQ ID NO:35), wherein Xaa₁ is any amino acid, Xaa₂ is Arg, Trp, 5-HTP,Tyr, or Phe, Xaa₃ is any amino acid, and n is 0, 1, 2, 3, 4, or 5. Thepolypeptide can inhibit the binding of FcRn to IgG Fc (e.g., Ile-253, orHis-435 in IgG4 or IgG1, IgG2, or IgG3 allotypes with His-435). Thepolypeptide can inhibit hydrophobic packing of FcRn with IgG Fc Met-252,Ile-253, Ser-254 His-435 and Tyr-436. The polypeptide can have a bindingaffinity of at least 1 μM (e.g., at least 100 nM or at least 10 nM) forthe C_(H)2-C_(H)3 cleft of an immmunoglobulin molecule having at leastone bound antigen. The amino-terminal amino acid of the polypeptide canbe acetylated. The carboxy-terminal amino acid of the polypeptide can beamidated. The polypeptide can contain entirely L-amino acids. At leastone amino acid of the polypeptide can be an unnatural amino acid, suchas 5-hydroxytrpophan (5-HTP). The polypeptide can be capable ofinhibiting the Fc-mediated formation of an immune complex. Thepolypeptide can be capable of inhibiting the binding of rheumatoidfactors to the C_(H)2-C_(H)3 cleft of an immunoglobulin molecule (e.g.,an immunoglobulin molecule bound by antigen). The polypeptide can becapable of inhibiting the binding of histones to the C_(H)2-C_(H)3 cleftof an immunoglobulin molecule (e.g., an immunoglobulin molecule bound byantigen). The polypeptide can be capable of inhibiting the binding ofFcR to the C_(H)2-C_(H)3 cleft of an immunoglobulin molecule (e.g., animmunoglobulin molecule bound by antigen). The polypeptide can becapable of inhibiting the binding of myelin basic protein to theC_(H)2-C_(H)3 cleft of an immunoglobulin molecule (e.g., animmunoglobulin molecule bound by antigen). The polypeptide can becapable of inhibiting the binding of pso p27 to the C_(H)2-C_(H)3 cleftof an immunoglobulin molecule (e.g., an immunoglobulin molecule bound byantigen) or to a rheumatoid factor. The polypeptide can be capable ofinhibiting the binding of Clq to the C_(H)2-C_(H)3 cleft of animmunoglobulin molecule (e.g., an immunoglobulin molecule bound byantigen). The polypeptide can have the amino acid sequence set forth inSEQ ID NO:16.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are three-dimensional, computer-generated structuralmodels of the C_(H)2-C_(H)3 cleft of an IgG molecule, showing theconformation of the cleft when the IgG is in an Fc-mediated immunecomplex or is non-immune complexed, as indicated.

FIG. 2A is a listing of atomic coordinates for an IgG molecule bound toa peptide ligand through the C_(H)2-C_(H)3 cleft. FIG. 2B is a listingof atomic coordinates for an IgG molecule bound to rheumatoid factorthrough the C_(H)2-C_(H)3 cleft.

FIG. 3 is a three-dimensional, computer-generated structural model of anIgG Fc C_(H)2-C_(H)3 cleft bound to a polypeptide having the amino acidsequence set forth in SEQ ID NO:5.

FIGS. 4A-4C are line graphs of arthritic indices in mice with or withoutcollagen-induced arthritis and treated or untreated as indicated. FIG.4A shows results for mice treated with the indicated amounts of ID 14polypeptide (SEQ ID NO:14). FIG. 4B shows results for mice treated withthe indicated amounts of ID 2 polypeptide (SEQ ID NO:2). FIG. 4C showsresults for mice treated with the indicated amounts of prednisolone orREMICADE®.

FIG. 5 is a three-dimensional structural model of interactions betweenFcRn and IgG Fc.

FIG. 6 is a three-dimensional structural model of interactions betweenIgG Fc and a polypeptide having the sequence set forth in SEQ ID NO:16,showing the hydrophobic packing with IgG Fc Met-252, Ile-253, Ser-254His435 and Tyr-436.

DETAILED DESCRIPTION

The invention provides polypeptides and other compounds capable ofinteracting with the C_(H)2-C_(H)3 cleft of an immunoglobulin molecule,such that interaction of the immunoglobulin with other molecules (e.g.,effectors or other immunoglobulins) is blocked. Methods for identifyingsuch polypeptides and other compounds also are provided, along withcompositions and articles of manufacture containing the polypeptides andcompounds. In addition, the invention provides methods for using thepolypeptides and compounds to inhibit immune complex formation and totreat diseases such as, for example, rheumatoid arthritis and systemiclupus erythematosus (SLE). These are described in the followingsubsections.

Immunoglobulins

The immunoglobulins make up a class of proteins found in plasma andother bodily fluids that exhibit antibody activity and bind to othermolecules (e.g., antigens and certain cell surface receptors) with ahigh degree of specificity. Based on their structure and biologicalactivity, immunoglobulins can be divided into five classes: IgM, IgG,IgA, IgD, and IgE. IgG is the most abundant antibody class in the body;this molecule assumes a twisted “Y” shape configuration. With theexception of the IgMs, immunoglobulins are composed mainly of fourpeptide chains that are linked by several intrachain and interchaindisulfide bonds. For example, the IgGs are composed of two polypeptideheavy chains (H chains) and two polypeptide light chains (L chains),which are coupled by disulfide bonds and non-covalent bonds to form aprotein molecule with a molecular weight of approximately 160,000daltons. The average IgG molecule contains approximately 4.5 interchaindisulfide bonds and approximately 12 intrachain disulfide bonds(Frangione and Milstein (1968) J. Mol. Biol. 33:893-906).

The light and heavy chains of immunoglobulin molecules are composed ofconstant regions and variable regions (see, e.g., Padlan (1994) Mol.Immunol. 31:169-217). For example, the light chains of an IgG1 moleculeeach contain a variable domain (V_(L)) and a constant domain (C_(L)).The heavy chains each have four domains: an amino terminal variabledomain (V_(H)), followed by three constant domains (C_(H)1, C_(H)2, andthe carboxy terminal C_(H)3). A hinge region corresponds to a flexiblejunction between the C_(H)1 and C_(H)2 domains. Papain digestion of anintact IgG molecule results in proteolytic cleavage at the hinge andproduces an Fc fragment that contains the C_(H)2 and C_(H)3 domains, andtwo identical Fab fragments that each contain a C_(H)1, C_(L), V_(H),and V_(L) domain. The Fc fragment has complement- and tissue-bindingactivity, while the Fab fragments have antigen-binding activity.

Immunoglobulin molecules can interact with other polypeptides throughvarious regions. The majority of antigen binding, for example, occursthrough the V_(L)/V_(H) region of the Fab fragment. The hinge regionalso is thought to be important, as immunological dogma states that thebinding sites for Fc receptors (FcR) are found in the hinge region ofIgG molecules (see, e.g., Raghavan and Bjorkman (1996) Annu. Rev. Dev.Biol. 12:181-200). More recent evidence, however, suggests that FcRinteracts with the hinge region primarily when the immunoglobulin ismonomeric (i.e., not immune-complexed). Such interactions typicallyinvolve the amino acids at positions 234-237 of the Ig molecule (Wienset al. (2000) J. Immunol. 164:5313-5318).

Immunoglobulin molecules also can interact with other polypeptidesthrough a cleft within the C_(H)2-C_(H)3 domain. The “C_(H)2-C_(H)3cleft” typically includes the amino acids at positions 251-255 withinthe C_(H)2 domain and the amino acids at positions 424-436 within theC_(H)3 domain. As used herein, numbering is with respect to an intactIgG molecule as in Kabat et al. (Sequences of Proteins of ImmunologicalInterest, 5^(th) ed., Public Health Service, U.S. Department of Healthand Human Services, Bethesda, Md.). Those of ordinary skill in the artcan readily determine the corresponding amino acids in otherimmunoglobulin classes.

The C_(H)2-C_(H)3 cleft is unusual in that it is characterized by both ahigh degree of solvent accessibility and a predominantly hydrophobiccharacter, suggesting that burial of an exposed hydrophobic surface isan important driving force behind binding at this site. Athree-dimensional change occurs at the IgG C_(H)2-C_(H)3 cleft uponantigen binding, allowing certain residues (e.g., a histidine atposition 435) to become exposed and available for binding. Directevidence of three-dimensional structural changes that occur upon antigenbinding was found in a study using monoclonal antibodies sensitive toconformational changes in the Fc region of human IgG. Five IgG epitopeswere altered by antigen binding: two within the hinge region and threewithin the C_(H)2-C_(H)3 cleft (Girkontraite et al. (1996) CancerBiother. Radiopharm. 11:87-96). Antigen binding therefore can beimportant for determining whether an immunoglobulin binds to othermolecules through the hinge or the Fc C_(H)2-C_(H)3 region.

The Fc region can bind to a number of effector molecules and otherproteins, including the following:

-   -   (1) FcRn—The neonatal Fc receptor determines the half life of        the antibody molecule in the general circulation (Leach et        al., (1996) J. Immunol. 157:3317-3322; Gheti and Ward (2000)        Ann. Rev. Immunol. 18:739-766). Mice genetically lacking FcRn        are protected from the deleterious effects of pathogenic        autoantibodies due to the shortened half-life of the        autoantibodies (Liu et al. (1997) J. Exp. Med. 186:777-783). An        inhibitor of FcRn binding to immune complexes or to pathogenic        autoantibodies would be useful in treating diseases involving        pathogenic autoantibodies and/or immune complexes.    -   (2) FcR—The cellular Fc Receptor provides a link between the        humoral immune response and cell-mediated effector systems        (Hamano et al. (2000) J. Immunol. 164:6113-6119; Coxon et        al. (2001) Immunity 14:693-704; Fossati et al. (2001) Eur. J.        Clin. Invest. 31:821-831). The Fcγ Receptors are specific for        IgG molecules, and include FcγRI, FcγRIa, FcγRIIb, and FcγRIII.        These isotypes bind with differing affinities to monomeric and        immune-complexed IgG.    -   (3) RF—Rheumatoid factors are immunoglobulins that bind to other        immune-complexed immunoglobulin molecules and can exacerbate        arthritis in animal models of rheumatoid arthritis (see, e.g.,        Ezaki et al. (1996) Clin. Exp. Immunol. 104:474-482).    -   (4) Histones—Histones are very basic, positively charged        proteins that bind to DNA and the negatively charged basement        membrane in the kidneys. In lupus nephritis, histones bind first        to the kidneys and then immune complexes bind to these        kidney-bound histones (Gussin et al. (2000) Clin. Immunol.        96:150-161).    -   (5) MBP—Myelin Basic Protein is the primary autoimmune target in        multiple sclerosis (MS; Sindic et al. (1980) Clin. Exp. Immunol.        41:1-7; Poston (1984) Lancet 1:1268-1271).    -   (6) Clq—The first component of the classical complement pathway        is Cl, which exists in blood serum as a complex of three        proteins, Clq, Clr, and Cls. The classical complement pathway is        activated when Clq binds to the Fc regions of antigen-bound IgG        or IgM. Although the binding of Clq to a single Fc region is        weak, Clq can form tight bonds to a cluster of Fc regions. At        this point Cl becomes proteolytically active.

The formation of immune complexes via interactions betweenimmunoglobulin Fc regions and other antibodies or other factors (e.g.,those described above) is referred to herein as “Fc-mediated immunecomplex formation” or “the Fc-mediated formation of an immune complex.”Immune complexes containing such interactions are termed “Fc-mediatedimmune complexes.” Fc-mediated immune complexes can includeimmunoglobulin molecules with or without bound antigen, and typicallyinclude C_(H)2-C_(H)3 cleft-specific ligands that have higher bindingaffinity for immune complexed antibodies than for monomeric antibodies.The large, generally insoluble complexes that can result fromFc-mediated immune complex formation typically are involved in thepathology of diseases such as, for example, RA and lupus nephritis.

Purified Polypeptides

As used herein, a “polypeptide” is any chain of amino acid residues,regardless of post-translational modification (e.g., phosphorylation orglycosylation). Polypeptides of the invention typically are between 10and 50 amino acids in length (e.g., 10, 11, 12, 13, 14, 15, 20, 25, 30,35, 40, 45, or 50 amino acids in length). Polypeptides of the inventionthat are between 10 and 20 amino acids in length (e.g., 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 amino acids in length) are particularlyuseful.

The amino acid sequences of the polypeptides provided herein aresomewhat constrained, but can have some variability. For example, thepolypeptides provided herein typically include the amino acid sequenceXaa₁-Cys-Ala-Xaa₂-His-Xaa₃-Xaa₄-Xaa₅-Leu-Val-Trp-Cys-Xaa₆ (SEQ ID NO:1),wherein the residues denoted by Xaa_(n) can display variability. Forexample, Xaa₁ can be absent or can be any amino acid (e.g., Arg or Asp).Xaa₂ can be Phe, Tyr, Trp, or Arg. Xaa₃ can be any amino acid. Xaa₄ canbe Gly or Ala, while Xaa₅ can be Glu or Ala. Like Xaa₁, Xaa₆ also can beabsent or can be any amino acid.

In one embodiment, a polypeptide can include the amino acid sequenceAsp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:2).Alternatively, a polypeptide can include the amino acid sequenceAsp-Cys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:3) orAsp-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:4). Inanother embodiment, a polypeptide can include the amino acid sequenceArg-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO: 5),Arg-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO: 6), orArg-Cys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:7).

In another embodiment, a polypeptide can include the amino acid sequenceCys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys (SEQ ID NO:8), in which Xaacan be Phe, Tyr, Trp, or Arg. For example, the invention providespolypeptides that include the following amino acid sequences:Cys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:9),Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:10), andCys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:11).

The polypeptides provided herein can be modified for use in vivo by theaddition, at the amino- or carboxy-terminal end, or at both ends, of astabilizing agent to facilitate survival of the polypeptide in vivo.This can be useful in situations in which peptide termini tend to bedegraded by proteases prior to cellular uptake. Such stabilizing groups(also referred to herein as blocking agents) can include, withoutlimitation, additional related or unrelated peptide sequences that canbe attached to the amino- and/or carboxy-terminal residues of thepolypeptide (e.g., an acetyl group attached to the N-terminal amino acidor an amide group attached to the C-terminal amino acid). Suchattachment can be achieved either chemically, during the synthesis ofthe polypeptide, or by recombinant DNA technology using methods familiarto those of ordinary skill in the art. Alternatively, blocking agentssuch as pyroglutamic acid or other molecules known in the art can beattached to the amino- and/or carboxy-terminal residues, or the aminogroup at the amino terminus or the carboxy group at the carboxy terminuscan be replaced with a different moiety.

In another embodiment, the polypeptides provided herein can be modifiedsuch that a stable protein is positioned at the amino terminus, at thecarboxy terminus, or both. Such a stablilizing group (also referred toas a “protein anchor”) typically is a small stable protein such as,without limitation, thioredoxin, glutathione sulfotransferase, maltosebinding protein, glutathione reductase, or a four-helix bundle proteinsuch as Rop protein, although no specific size limitation on the proteinanchor is intended.

Proteins suitable for use as stabilizing groups can be either naturallyoccurring or non-naturally occurring. Such stabilizing groups can beisolated from an endogenous source, chemically or enzymaticallysynthesized, or produced using recombinant DNA technology. Proteins thatare particularly well suited for use as stabilizing groups are thosethat are relatively short in length and form very stable structures insolution. Proteins having molecular weights of less than about 70 kD(e.g., less than about 65, 60, 50, 40, 25, or 12 kD) can be particularlyuseful as stabilizing groups. For example, human serum albumin has amolecular weight of about 64 kD; E. coli thioredoxin has a molecularweight of about 11.7 kD; E. coli glutathione sulfotransferase has amolecular weight of about 22.9 kD; Rop from the ColEl replicon has amolecular weight of about 7.2 kD; and maltose binding protein (withoutits signal sequence) has a molecular weight of about 40.7 kD. The smallsize of the Rop protein makes it especially useful as a stabilizinggroup, since it is less likely than larger proteins to interfere withaccessibility of the linked peptide, thus preserving its bioactivity.Rop's highly ordered anti-parallel four-helix bundle topology (afterdimerization), slow unfolding kinetics (see, e.g., Betz et al. (1997)Biochem. 36:2450-2458), and lack of disulfide bonds also contribute toits usefulness as a peptide anchor according to the invention. Otherproteins with similar folding kinetics and/or thermodynamic stability(e.g., Rop has a midpoint temperature of denaturation (Tm) of about 71°C.; Steif et al. (1993) Biochem. 32:3867-3876) also are useful asstabilizing groups. Peptides or proteins having highly stable tertiarymotifs, such as a four-helix bundle topology, are particularly useful.

In another embodiment, a stabilizing group such as a proline, a Pro-Prosequence, or an Xaa-Pro-Pro sequence (e.g., Ala-Pro-Pro) can bepositioned at the amino terminus of a polypeptide (see, e.g., WO00/22112). For example, a polypeptide can include the amino acidsequence Xaa₁-Pro-Pro-Cys-Ala-Xaa₂-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr(SEQ ID NO: 12), where Xaa₁ is any amino acid (e.g., Ala), and Xaa₂ isTrp, Tyr, Phe, or Arg. For example, a polypeptide can include the aminoacid sequenceXaa₁-Pro-Pro-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ IDNO:13), Xaa₁-Pro-Pro-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr(SEQ ID NO:14), orXaa₁-Pro-Pro-Cys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:15). Alternatively, a polypeptide can include the amino acid sequenceXaa₁-Pro-Pro-Asp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ IDNO:16), Xaa₁-Pro-Pro-Asp-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr(SEQ ID NO:17),Xaa₁-Pro-Pro-Asp-Cys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ IDNO:18), Xaa₁-Pro-Pro-Arg-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr(SEQ ID NO:19),Xaa₁-Pro-Pro-Arg-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ IDNO:20), orXaa₁-Pro-Pro-Arg-Cys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ IDNO: 21).

Alternatively, the polypeptides provided herein can have a proline, aPro-Pro sequence, or a Pro-Pro-Xaa sequence (e.g., Pro-Pro-Ala)positioned at their carboxy termini. For example, a polypeptide caninclude the amino acid sequenceCys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Pro-Pro-Xaa (SEQ IDNO:22), Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Pro-Pro-Xaa (SEQID NO:23), Cys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Pro-Pro-Xaa(SEQ ID NO: 24,Asp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Pro-Pro-Xaa (SEQ IDNO:25), Asp-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Pro-Pro-Xaa(SEQ ID NO:26),Asp-Cys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Pro-Pro-Xaa (SEQ IDNO:27), Arg-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Pro-Pro-Xaa(SEQ ID NO:28),Arg-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Pro-Pro-Xaa (SEQ IDNO:29), orArg-Cys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Pro-Pro-Xaa (SEQ IDNO:30), wherein Xaa can be any amino acid. In one embodiment, apolypeptide can have both an Xaa-Pro-Pro (e.g., Ala-Pro-Pro) sequence atits amino termini and a Pro-Pro-Xaa (e.g., Pro-Pro-Ala) sequence at itscarboxy terminus.

The polypeptides provided herein also can include additional amino acidsequences at the amino terminus of the sequence set forth in SEQ IDNO:1, the carboxy terminus of the sequence set forth in SEQ ID NO:1, orboth. For example, a polypeptide can contain the amino acid sequenceTrp-Glu-Ala-Xaa₁-Cys-Ala-Xaa₂-His-Xaa₃-Xaa₄-Xaa₅-Leu-Val-Trp-Cys-Xaa₆-Lys-Val-Glu-Glu(SEQ ID NO:31), wherein the residues denoted by Xaa_(n) can displayvariability. As for the amino acid sequence set forth in SEQ ID NO:1,Xaa₁ can be absent or can be any amino acid (e.g., Arg or Asp); Xaa₂ canbe Phe, Tyr, Trp, or Arg; Xaa₃ can be any amino acid; Xaa₄ can be Gly orAla; Xaa₅ can be Glu or Ala; and Xaa₆ can be absent or can be any aminoacid. In one embodiment, a polypeptide can include the amino acidsequenceTrp-Glu-Ala-Asp-Cys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Lys-Val-Glu-Glu(SEQ ID NO:32), where Xaa is Arg, Trp, Tyr, or Phe. For example, apolypeptide can include the amino acid sequenceTrp-Glu-Ala-Asp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Lys-Val-Glu-Glu(SEQ ID NO:33).

In another embodiment, a polypeptide can consist of the amino acidsequence(Xaa₁)_(n)-Xaa₂-Cys-Ala-Xaa₃-His-Xaa₄-Xaa₅-Xaa₆-Leu-Val-Trp-Cys-(Xaa₇)_(n)(SEQ ID NO:34), wherein the residues denoted by Xaa can displayvariability, and n can be an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10). For example, Xaa₁ can be any amino acid; Xaa₂ canbe absent or can be any amino acid (e.g., Arg or Asp); Xaa₃ can be Phe,Tyr, Trp, Arg, or 5-HTP; Xaa₄ can be any amino acid; Xaa₅ can be Gly orAla; Xaa₆ can be Glu or Ala; Xaa₇ can be any amino acid; and n can befrom 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5). Alternatively, a polypeptideconsist of the amino acid sequence(Xaa₁)_(n)-Cys-Ala-Xaa₂-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-(Xaa₃)_(n)(SEQ ID NO:35), wherein Xaa₁ is any amino acid, Xaa₂ is Tyr, Trp, Phe,Arg, or 5-HTP, Xaa₃ is any amino acid, and n is an integer from 0 to 5(e.g., 0, 1, 2, 3, 4, or 5). Examples of polypeptides within theseembodiments include, without limitation, polypeptides consisting of theamino acid sequenceAla-Pro-Pro-Leu-Asp-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Ala-Leu-Pro-Pro-Ala(SEQ ID NO:36),Ala-Ala-Arg-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Ala-Ala (SEQID NO:37), orAla-Pro-Pro-Asp-Cys-Ala-Phe-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Ala-Ala(SEQ ID NO:38).

The amino acid sequences set forth in SEQ ID NOs:1-38 typically containtwo cysteine residues. Polypeptides containing these amino acidsequences typically cyclize due to formation of a disulfide bond betweenthe two cysteine residues. A person having ordinary skill in the artcan, for example, use Ellman's Reagent to determine whether a peptidecontaining multiple cysteine residues is cyclized. In some embodiments,these cysteine residues can be substituted with other natural ornon-natural amino acid residues that can form lactam bonds rather thandisulfide bonds. For example, one cysteine residue could be replacedwith aspartic acid or glutamic acid, while the other could be replacedwith ornithine or lysine. Any of these combinations could yield a lactambridge. By varying the amino acids that form a lactam bridge, apolypeptide provided herein can be generated that contains a bridgeapproximately equal in length to the disulfide bond that would be formedif two cysteine residues were present in the polypeptide.

The polypeptides provided herein can contain an amino acid tag. A “tag”is generally a short amino acid sequence that provides a ready means ofdetection or purification through interactions with an antibody againstthe tag or through other compounds or molecules that recognize the tag.For example, tags such as c-myc, hemagglutinin, polyhistidine, or FLAG®can be used to aid purification and detection of a polypeptide. As anexample, a polypeptide with a polyhistidine tag can be purified based onthe affinity of histidine residues for nickel ions (e.g., on a Ni-NTAcolumn), and can be detected in western blots by an antibody againstpolyhistidine (e.g., the Penta-His antibody; Qiagen, Valencia, Calif.).Tags can be inserted anywhere within the polypeptide sequence, althoughinsertion at the amino- or carboxy-terminus is particularly useful.

The term “amino acid” refers to natural amino acids, unnatural aminoacids, and amino acid analogs, all in their D and L stereoisomers iftheir structures so allow. Natural amino acids include alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan (Trp), tyrosine (Tyr), and valine (Val). Unnatural aminoacids include, but are not limited to azetidinecarboxylic acid,2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionicacid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid,2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid,2-aminopimelic acid, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,4-hydroxyproline, 5-hydroxytryptophan, isodesmosine, allo-isoleucine,N-methylglycine, N-methylisoleucine, N-methylvaline, norvaline,norleucine, ornithine, and pipecolic acid.

An “analog” is a chemical compound that is structurally similar toanother but differs slightly in composition (as in the replacement ofone atom by an atom of a different element or in the presence of aparticular functional group). An “amino acid analog” therefore isstructurally similar to a naturally occurring amino acid molecule as istypically found in native polypeptides, but differs in composition suchthat either the C-terminal carboxy group, the N-terminal amino group, orthe side-chain functional group has been chemically modified to anotherfunctional group. Amino acid analogs include natural and unnatural aminoacids which are chemically blocked, reversibly or irreversibly, ormodified on their N-terminal amino group or their side-chain groups, andinclude, for example, methionine sulfoxide, methionine sulfone,S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide andS-(carboxymethyl)-cysteine sulfone. Amino acid analogs may be naturallyoccurring, or can be synthetically prepared. Non-limiting examples ofamino acid analogs include aspartic acid-(beta-methyl ester), an analogof aspartic acid; N-ethylglycine, an analog of glycine; and alaninecarboxamide, an analog of alanine. Other examples of amino acids andamino acids analogs are listed in Gross and Meienhofer, The Peptides:Analysis, Synthesis, Biology, Academic Press, Inc., New York (1983).

The stereochemistry of a polypeptide can be described in terms of thetopochemical arrangement of the side chains of the amino acid residuesabout the polypeptide backbone, which is defined by the peptide bondsbetween the amino acid residues and the α-carbon atoms of the bondedresidues. In addition, polypeptide backbones have distinct termini andthus direction. The majority of naturally occurring amino acids areL-amino acids. Naturally occurring polypeptides are largely comprised ofL-amino acids.

D-amino acids are the enantiomers of L-amino acids and can form peptidesthat are herein referred to as “inverso” polypeptides (i.e., peptidescorresponding to native peptides but made up of D-amino acids ratherthan L-amino acids). A “retro” polypeptide is made up of L-amino acids,but has an amino acid sequence in which the amino acid residues areassembled in the opposite direction of the native peptide sequence.

“Retro-inverso” modification of naturally occurring polypeptidesinvolves the synthetic assembly of amino acids with a-carbonstereochemistry opposite to that of the corresponding L-amino acids(i.e., D- or D-allo-amino acids), in reverse order with respect to thenative polypeptide sequence. A retro-inverso analog thus has reversedtermini and reversed direction of peptide bonds, while approximatelymaintaining the topology of the side chains as in the native peptidesequence. The term “native” refers to any sequence of L-amino acids usedas a starting sequence for the preparation of partial or complete retro,inverso or retro-inverso analogs.

Partial retro-inverso polypeptide analogs are polypeptides in which onlypart of the sequence is reversed and replaced with enantiomeric aminoacid residues. Since the retro-inverted portion of such an analog hasreversed amino and carboxyl termini, the amino acid residues flankingthe retro-inverted portion can be replaced by side-chain-analogousα-substituted geminal-diaminomethanes and malonates, respectively.Alternatively, a polypeptide can be a complete retro-inverso analog, inwhich the entire sequence is reversed and replaced with D-amino acids.

The invention also provides peptidomimetic compounds that are designedon the basis of the amino acid sequences of polypeptides. Peptidomimeticcompounds are synthetic, non-peptide compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide, and can thus confer the same or similar function asthe selected peptide. Peptidomimetic compounds of the invention can bedesigned to mimic any of the polypeptides of the invention.

Peptidomimetic compounds that are protease resistant are particularlyuseful. Furthermore, peptidomimetic compounds may have additionalcharacteristics that enhance therapeutic utility, such as increased cellpermeability and prolonged biological half-life. Such compoundstypically have a backbone that is partially or completely non-peptide,but with side groups that are identical or similar to the side groups ofthe amino acid residues that occur in the peptide upon which thepeptidomimetic compound is based. Several types of chemical bonds (e.g.,ester, thioester, thioamide, retroamide, reduced carbonyl, dimethyleneand ketomethylene) are known in the art to be useful substitutes forpeptide bonds in the construction of peptidomimetic compounds.

The interactions between a polypeptide of the invention and animmunoglobulin molecule typically occur through the C_(H)2-C_(H)3 cleftof the immunoglobulin. Such interactions are engendered through physicalproximity and are mediated by, for example, hydrophobic interactions.The “binding affinity” of a polypeptide for an immunoglobulin moleculerefers to the strength of the interaction between the polypeptide andthe immunoglobulin. Binding affinity typically is expressed as anequilibrium dissociation constant (K_(d)), which is calculated asK_(d)=k_(off)/k_(on), where k_(off)=the kinetic dissociation constant ofthe reaction, and k_(on)=the kinetic association constant of thereaction. K_(d) is expressed as a concentration, with a low K_(d) value(e.g., less than 100 nM) signifying high affinity. Polypeptides of theinvention that can interact with an immunoglobulin molecule typicallyhave a binding affinity of at least 1 μM (e.g., at least 500 nM, atleast 100 nM, at least 50 nM, or at least 10 nM) for the C_(H)2-C_(H)3cleft of the immunoglobulin.

Polypeptides provided herein can bind with substantially equivalentaffinity to immunoglobulin molecules that are bound by antigen and tomonomeric immunoglobulins. Alternatively, polypeptides of the inventioncan have a higher binding affinity (e.g., at least 10-fold, at least100-fold, or at least 1000-fold higher binding affinity) forimmunoglobulin molecules that are bound by antigen than for monomericimmunoglobulins. Conformational changes that occur within the Fc regionof an immunoglobulin molecule upon antigen binding to the Fab region arelikely involved in a difference in affinity. The crystal structures ofbound and unbound NC6.8 Fab (from a murine monoclonal antibody) showedthat the tail of the Fab heavy chain was displaced by 19 angstroms incrystals of the antigen/antibody complex, as compared to its position inunbound Fab (Guddat et al. (1994) J. Mol. Biol. 236-247-274). Since theC-terminal tail of the Fab region is connected to the Fc region in anintact antibody, this shift would be expected to affect the conformationof the C_(H)2-C_(H)3 cleft. Furthermore, examination of severalthree-dimensional structures of intact immunoglobulins revealed a directphysical connection between the Fab heavy chain and the Fc C_(H)2-C_(H)3cleft (Harris et al. (1997) Biochemistry 36:1581-1597; Saphire et al.(2001) Science 293:1155-1159).

Molecular modeling of the C_(H)2-C_(H)3 cleft of monomeric (i.e.,unbound) and immune-complexed IgG (see FIGS. 1A and 1B) revealed thatthe monomeric Fc C_(H)2-C_(H)3 cleft has a closed configuration, whichcan prevent binding to critical amino acid residues (e.g., His435; see,for example, O'Brien et al. (1994) Arch. Biochem. Biophys. 310:25-31;Jefferies et al. (1984) Immunol. Lett. 7:191-194; and West et al. (2000)Biochemistry 39:9698-9708). Immune-complexed (antigen-bound) IgG,however, has a more open configuration and thus is more conducive toligand binding. The binding affinity of RF for immune-complexed IgG, forexample, is much greater than the binding affinity of RF for monomericIgG (Corper et al. (1997) Nat. Struct. Biol. 4:374; Sohi et al. (1996)Immunol. 88:636). The same typically is true for polypeptides of theinvention.

Because polypeptides of the invention can bind to the C_(H)2-C_(H)3cleft of immunoglobulin molecules, they are useful for blocking theinteraction of other factors (e.g., FcRn, FcR, RF, histones, MBP, andother immunoglobulins) to the Fc region of the immunoglobulin, and thuscan inhibit Fc-mediated immune complex formation. By “inhibit” is meantthat Fc-mediated immune complex formation is reduced in the presence ofa polypeptide of the invention, as compared to the level of immunecomplex formation in the absence of the polypeptide. Such inhibiting canoccur in vitro (e.g., in a test tube) or in vivo (e.g., in anindividual). Any suitable method can be used to assess the level ofimmune complex formation. Many such methods are known in the art, andsome of these are described herein.

Polypeptides of the invention typically interact with the C_(H)2-C_(H)3cleft of an immunoglobulin molecule in a monomeric fashion (i.e.,interact with only one immunoglobulin molecule and thus do not link twoor more immunoglobulin molecules together). Interactions with otherimmunoglobulin molecules through the Fc region therefore are precludedby the presence of the polypeptide. The inhibition of Fc-mediated immunecomplex formation can be assessed in vitro, for example, by incubatingan IgG molecule with a labeled immunoglobulin molecule (e.g., afluorescently labeled RF molecule) in the presence and absence of apolypeptide of the invention, and measuring the amount of labeledimmunoglobulin that is incorporated into an immune complex. Othermethods suitable for detecting immune complex formation also may beused, as discussed below.

Preparation and Purification of Polypeptides

Polypeptides of the invention can be produced by a number of methods,many of which are well known in the art. By way of example and notlimitation, a polypeptide can be obtained by extraction from a naturalsource (e.g., from isolated cells, tissues or bodily fluids), byexpression of a recombinant nucleic acid encoding the polypeptide (as,for example, described below), or by chemical synthesis (e.g., bysolid-phase synthesis or other methods well known in the art, includingsynthesis with an ABI peptide synthesizer; Applied Biosystems, FosterCity, Calif.). Methods for synthesizing retro-inverso polypeptideanalogs (Bonelli et al. (1984) Int. J. Peptide Protein Res. 24:553-556;and Verdini and Viscomi (1985) J. Chem. Soc. Perkin Trans. I:697-701),and some processes for the solid-phase synthesis of partialretro-inverso peptide analogs also have been described (see, forexample, European Patent number EP0097994).

The invention provides isolated nucleic acid molecules encoding thepolypeptides described herein. As used herein, “nucleic acid” refers toboth RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g.,chemically synthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded (i.e., a sense or an antisense single strand). The term“isolated” as used herein with reference to a nucleic acid refers to anaturally-occurring nucleic acid that is not immediately contiguous withboth of the sequences with which it is immediately contiguous (one atthe 5′ end and one at the 3′ end) in the naturally-occurring genome ofthe organism from which it is derived. The term “isolated” as usedherein with respect to nucleic acids also includes anynon-naturally-occurring nucleic acid sequence, since suchnon-naturally-occurring sequences are not found in nature and do nothave immediately contiguous sequences in a naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences that is normally immediatelycontiguous with the DNA molecule in a naturally-occurring genome isremoved or absent. Thus, an isolated nucleic acid includes, withoutlimitation, a DNA molecule that exists as a separate molecule (e.g., achemically synthesized nucleic acid, or a cDNA or genomic DNA fragmentproduced by PCR or restriction endonuclease treatment) independent ofother sequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a recombinant DNA moleculethat is part of a hybrid or fusion nucleic acid. A nucleic acid existingamong hundreds to millions of other nucleic acids within, for example,cDNA libraries or genomic libraries, or gel slices containing a genomicDNA restriction digest, is not considered an isolated nucleic acid.

The invention also provides vectors containing the nucleic acidsdescribed herein. As used herein, a “vector” is a replicon, such as aplasmid, phage, or cosmid, into which another DNA segment may beinserted so as to bring about the replication of the inserted segment.The vectors of the invention are preferably expression vectors, in whichthe nucleotides encode the polypeptides of the invention with aninitiator methionine, operably linked to expression control sequences.As used herein, “operably linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest. An “expression controlsequence” is a DNA sequence that controls and regulates thetranscription and translation of another DNA sequence, and an“expression vector” is a vector that includes expression controlsequences, so that a relevant DNA segment incorporated into the vectoris transcribed and translated. A coding sequence is “operably linked”and “under the control” of transcriptional and translational controlsequences in a cell when RNA polymerase transcribes the coding sequenceinto mRNA, which then is translated into the protein encoded by thecoding sequence.

Methods well known to those skilled in the art may be used to subcloneisolated nucleic acid molecules encoding polypeptides of interest intoexpression vectors containing relevant coding sequences and appropriatetranscriptional/translational control signals. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual (2^(nd)edition), Cold Spring Harbor Laboratory, New York (1989); and Ausubel etal., Current Protocols in Molecular Biology, Green Publishing Associatesand Wiley Interscience, New York (1989). Expression vectors of theinvention can be used in a variety of systems (e.g., bacteria, yeast,insect cells, and mammalian cells), as described herein. Examples ofsuitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, herpes viruses, retroviruses,vaccinia viruses, adenoviruses, and adeno-associated viruses. A widevariety of suitable expression vectors and systems are commerciallyavailable, including the pET series of bacterial expression vectors(Novagen, Madison, Wis.), the Adeno-X expression system (Clontech), theBaculogold baculovirus expression system (BD Biosciences Pharmingen, SanDiego, Calif.), and the pCMV-Tag vectors (Stratagene, La Jolla, Calif.).

Expression vectors that encode the polypeptides of the invention can beused to produce the polypeptides. Expression systems that can be usedfor small or large scale production of the polypeptide of the inventioninclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA, or cosmid DNA expression vectors containing the nucleicacid molecules of the invention; yeast (e.g., S. cerevisiae) transformedwith recombinant yeast expression vectors containing the nucleic acidmolecules of the invention; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing thenucleic acid molecules of the invention; plant cell systems infectedwith recombinant virus expression vectors (e.g., tobacco mosaic virus)or transformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the nucleic acid molecules of the invention; ormammalian cell systems (e.g., primary cells or immortalized cell linessuch as COS cells, CHO cells, HeLa cells, HEK 293 cells, and 3T3 L1cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., the metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoterand the cytomegalovirus promoter), along with the nucleic acids of theinvention.

The term “purified polypeptide” as used herein refers to a polypeptidethat either has no naturally occurring counterpart (e.g., apeptidomimetic), or has been chemically synthesized and is thusuncontaminated by other polypeptides, or that has been separated orpurified from other cellular components by which it is naturallyaccompanied (e.g., other cellular proteins, polynucleotides, or cellularcomponents). Typically, the polypeptide is considered “purified” when itis at least 70%, by dry weight, free from the proteins and naturallyoccurring organic molecules with which it naturally associates. Apreparation of the purified polypeptide of the invention therefore canbe, for example, at least 80%, at least 90%, or at least 99%, by dryweight, the polypeptide of the invention. Suitable methods for purifyingthe polypeptides of the invention can include, for example, affinitychromatography, immunoprecipitation, size exclusion chromatography, andion exchange chromatography. The extent of purification can be measuredby any appropriate method, including but not limited to: columnchromatography, polyacrylamide gel electrophoresis, or high-performanceliquid chromatography.

Methods of Modeling, Designing, and Identifying Compounds

The invention provides methods for designing, modeling, and identifyingcompounds that can bind to the C_(H)2-C_(H)3 cleft of an immunoglobulinmolecule and thus serve as inhibitors of Fc-mediated immune complexformation. Such compounds also are referred to herein as “ligands.”Compounds designed, modeled, and identified by methods of the inventiontypically can interact with an immunoglobulin molecule through theC_(H)2-C_(H)3 cleft, and typically have a binding affinity of at least 1μM (e.g., at least 500 nM, at least 100 nM, at least 50 nM, or at least10 nM) for the C_(H)2-C_(H)3 cleft of the immunoglobulin. Such compoundsgenerally have higher binding affinity (e.g., at least 10-fold, at least100-fold, or at least 1000-fold higher binding affinity) forimmune-complexed immunoglobulin molecules than for monomericimmunoglobulin molecules.

Compounds of the invention typically interact with the C_(H)2-C_(H)3cleft of an immunoglobulin molecule in a monomeric fashion (i.e.,interact with only one immunoglobulin molecule and thus do not link twoor more immunoglobulin molecules together). The interactions between acompound and an immunoglobulin molecule typically involve the amino acidresidues at positions 252, 253, 435, and 436 of the immunoglobulin(number according to Kabat, supra). The interaction between compounds ofthe invention and the C_(H)2-C_(H)3 cleft renders the compounds capableof inhibiting the Fc-mediated formation of immune complexes by blockingthe binding of other factors (e.g., RF, histones, FcR, FcRn, Clq, MBP,and psoriasis associated antigen pso p27) to the C_(H)2-C_(H)3 cleft.

Compounds identified by methods of the invention can be polypeptidessuch as, for example, those described herein. Alternatively, a compoundcan be any suitable type of molecule that can specifically bind to theC_(H)2-C_(H)3 cleft of an immunoglobulin molecule. Compounds such asquercetin, boswellic acids, and statins are particularly useful.

By “modeling” is meant quantitative and/or qualitative analysis ofreceptor-ligand structure/function based on three-dimensional structuralinformation and receptor-ligand interaction models. This includesconventional numeric-based molecular dynamic and energy minimizationmodels, interactive computer graphic models, modified molecularmechanics models, distance geometry and other structure-based constraintmodels. Modeling typically is performed using a computer and may befurther optimized using known methods.

Methods of designing ligands that bind specifically (i.e., with highaffinity) to the C_(H)2-C_(H)3 cleft of an immunoglobulin moleculehaving bound antigen typically are computer-based, and involve the useof a computer having a program capable of generating an atomic model.Computer programs that use X-ray crystallography data are particularlyuseful for designing ligands that can interact with an Fc C_(H)2-C_(H)3cleft. Programs such as RasMol, for example, can be used to generate athree dimensional model of a C_(H)2-C_(H)3 cleft and/or determine thestructures involved in ligand binding. Computer programs such as INSIGHT(Accelrys, Burlington, Mass.), GRASP (Anthony Nicholls, ColumbiaUniversity), Dock (Molecular Design Institute, University of Californiaat San Francisco), and Auto-Dock (Accelrys) allow for furthermanipulation and the ability to introduce new structures.

Methods of the invention can include, for example, providing to acomputer the atomic structural coordinates (e.g., the coordinates shownin FIGS. 2A and 2B) for amino acid residues within the C_(H)2-C_(H)3cleft (e.g., amino acid residues at positions 252, 253, 435, and 436 ofthe cleft) of an immunoglobulin molecule in an Fc-mediated immunecomplex, using the computer to generate an atomic model of theC_(H)2-C_(H)3 cleft, further providing the atomic structural coordinatesof a candidate compound and generating an atomic model of the compoundoptimally positioned within the C_(H)2-C_(H)3 cleft, and identifying thecandidate compound as a ligand of interest if the compound interactswith the amino acid residues at positions 252, 253, 435, and 436 of thecleft. The data provided to the computer also can include the atomiccoordinates of amino acid residues at positions in addition to 252, 253,435, and 436. By “optimally positioned” is meant positioned to optimizehydrophobic interactions between the candidate compound and the aminoacid residues at positions 252, 253, 435, and 436 of the C_(H)2-C_(H)3cleft.

Alternatively, a method for designing a ligand having specific bindingaffinity for the C_(H)2-C_(H)3 cleft of an immunoglobulin molecule canutilize a computer with an atomic model of the cleft stored in itsmemory. The atomic coordinates of a candidate compound then can beprovided to the computer, and an atomic model of the candidate compoundoptimally positioned can be generated. As described herein, a candidatecompound can be identified as a ligand having specific binding affinityfor the C_(H)2-C_(H)3 cleft of an immunoglobulin molecule if, forexample, the compound interacts with the amino acid residues atpositions 252, 253, 435, and 436 of the cleft.

Compounds of the invention also may be interactively designed fromstructural information of the compounds described herein using otherstructure-based design/modeling techniques (see, e.g., Jackson (1997)Seminars in Oncology 24:L164-172; and Jones et al. (1996) J. Med. Chem.39:904-917).

Compounds and polypeptides of the invention also can be identified by,for example, identifying candidate compounds by computer modeling asfitting spatially and preferentially (i.e., with high affinity) into theC_(H)2-C_(H)3 cleft of an immunoglobulin molecule, and then screeningthose compounds in vitro or in vivo for the ability to inhibitFc-mediated immune complex formation. Suitable methods for such in vitroand in vivo screening include those described herein.

Compositions and Articles of Manufacture

The invention provides methods for treating conditions that arise fromabnormal Fc-mediated immune complex formation (e.g., over-production ofFc-mediated immune complexes). By these methods, polypeptides andcompounds in accordance with the invention are administered to a subject(e.g., a human or another mammal) having a disease or disorder (e.g.,rheumatoid arthritis) that can be alleviated by modulating Fc-mediatedimmune complex formation. Typically, one or more polypeptides orcompounds can be administered to a subject suspected of having a diseaseor condition associated with immune complex formation.

The polypeptides and compounds provided herein can be used in themanufacture of a medicament (i.e., a composition) for treatingconditions that arise from abnormal Fc-mediated immune complexformation. Compositions of the invention typically contain one or morepolypeptides and compounds described herein. A C_(H)2-C_(H)3 bindingpolypeptide, for example, can be in a pharmaceutically acceptablecarrier or diluent, and can be administered in amounts and for periodsof time that will vary depending upon the nature of the particulardisease, its severity, and the subject's overall condition. Typically,the polypeptide is administered in an inhibitory amount (i.e., in anamount that is effective for inhibiting the production of immunecomplexes in the cells or tissues contacted by the polypeptide). Thepolypeptide and methods of the invention also can be usedprophylactically, e.g., to minimize immunoreactivity in a subject atrisk for abnormal or over-production of immune complexes (e.g., atransplant recipient).

The ability of a polypeptide to inhibit Fc-mediated immune complexformation can be assessed by, for example, measuring immune complexlevels in a subject before and after treatment. A number of methods canbe used to measure immune complex levels in tissues or biologicalsamples, including those that are well known in the art. If the subjectis a research animal, for example, immune complex levels in the jointscan be assessed by immunostaining following euthanasia. Theeffectiveness of an inhibitory polypeptide also can be assessed bydirect methods such as measuring the level of circulating immunecomplexes in serum samples. Alternatively, indirect methods can be usedto evaluate the effectiveness of polypeptides in live subjects. Forexample, reduced immune complex formation can be inferred from reducedpain in rheumatoid arthritis patients. Animal models also can be used tostudy the development of and relief from conditions such as rheumatoidarthritis.

Methods for formulating and subsequently administering therapeuticcompositions are well known to those skilled in the art. Dosing isgenerally dependent on the severity and responsiveness of the diseasestate to be treated, with the course of treatment lasting from severaldays to several months, or until a cure is effected or a diminution ofthe disease state is achieved. Persons of ordinary skill in the artroutinely determine optimum dosages, dosing methodologies and repetitionrates. Optimum dosages can vary depending on the relative potency ofindividual polypeptides, and can generally be estimated based on EC50found to be effective in in vitro and in vivo animal models. Typically,dosage is from 0.01 μg to 100 g per kg of body weight, and may be givenonce or more daily, biweekly, weekly, monthly, or even less often.Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate.

The present invention provides pharmaceutical compositions andformulations that include the polypeptides and/or compounds of theinvention. Polypeptides therefore can be admixed, encapsulated,conjugated or otherwise associated with other molecules, molecularstructures, or mixtures of compounds such as, for example, liposomes,polyethylene glycol, receptor targeted molecules, or oral, rectal,topical or other formulations, for assisting in uptake, distributionand/or absorption.

A “pharmaceutically acceptable carrier” (also referred to herein as an“excipient”) is a pharmaceutically acceptable solvent, suspending agent,or any other pharmacologically inert vehicle for delivering one or moretherapeutic compounds (e.g., C_(H)2-C_(H)3 binding polypeptides) to asubject. Pharmaceutically acceptable carriers can be liquid or solid,and can be selected with the planned manner of administration in mind soas to provide for the desired bulk, consistency, and other pertinenttransport and chemical properties, when combined with one or more oftherapeutic compounds and any other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers that do notdeleteriously react with amino acids include, by way of example and notlimitation: water; saline solution; binding agents (e.g.,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose and other sugars, gelatin, or calcium sulfate); lubricants(e.g., starch, polyethylene glycol, or sodium acetate); disintegrates(e.g., starch or sodium starch glycolate); and wetting agents (e.g.,sodium lauryl sulfate).

The pharmaceutical compositions of the present invention can beadministered by a number of methods, depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be, for example, topical (e.g., transdermal,sublingual, ophthalmic, or intranasal); pulmonary (e.g., by inhalationor insufflation of powders or aerosols); oral; or parenteral (e.g., bysubcutaneous, intrathecal, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip). Administration canbe rapid (e.g., by injection) or can occur over a period of time (e.g.,by slow infusion or administration of slow release formulations). Fortreating tissues in the central nervous system, C_(H)2-C_(H)3 bindingpolypeptides can be administered by injection or infusion into thecerebrospinal fluid, preferably with one or more agents capable ofpromoting penetration of the polypeptides across the blood-brainbarrier.

Formulations for topical administration of C_(H)2-C_(H)3 bindingpolypeptides include, for example, sterile and non-sterile aqueoussolutions, non-aqueous solutions in common solvents such as alcohols, orsolutions in liquid or solid oil bases. Such solutions also can containbuffers, diluents and other suitable additives. Pharmaceuticalcompositions and formulations for topical administration can includetransdermal patches, ointments, lotions, creams, gels, drops,suppositories, sprays, liquids, and powders. Nasal sprays areparticularly useful, and can be administered by, for example, anebulizer or another nasal spray device. Administration by an inhaleralso is particularly useful. Conventional pharmaceutical carriers,aqueous, powder or oily bases, thickeners and the like may be necessaryor desirable.

Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, or tablets. Such compositions alsocan incorporate thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders.

Compositions and formulations for parenteral, intrathecal orintraventricular administration can include sterile aqueous solutions,which also can contain buffers, diluents and other suitable additives(e.g., penetration enhancers, carrier compounds and otherpharmaceutically acceptable carriers).

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, aqueous suspensions, andliposome-containing formulations. These compositions can be generatedfrom a variety of components that include, for example, preformedliquids, self-emulsifying solids and self-emulsifying semisolids.Emulsions are often biphasic systems comprising of two immiscible liquidphases intimately mixed and dispersed with each other; in general,emulsions are either of the water-in-oil (w/o) or oil-in-water (o/w)variety. Emulsion formulations have been widely used for oral deliveryof therapeutics due to their ease of formulation and efficacy ofsolubilization, absorption, and bioavailability.

Liposomes are vesicles that have a membrane formed from a lipophilicmaterial and an aqueous interior that can contain the composition to bedelivered. Liposomes can be particularly useful due to their specificityand the duration of action they offer from the standpoint of drugdelivery. Liposome compositions can be formed, for example, fromphosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoylphosphatidylethanolamine. Numerous lipophilic agents are commerciallyavailable, including LIPOFECTIN® (Invitrogen/Life Technologies,Carlsbad, Calif.) and EFFECTENE™ (Qiagen, Valencia, Calif.).

Polypeptides of the invention further encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the invention providespharmaceutically acceptable salts of polypeptides, prodrugs andpharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. The term “prodrug” indicates a therapeutic agent that isprepared in an inactive form and is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes or other chemicals and/or conditions. The term “pharmaceuticallyacceptable salts” refers to physiologically and pharmaceuticallyacceptable salts of the polypeptides of the invention (i.e., salts thatretain the desired biological activity of the parent polypeptide withoutimparting undesired toxicological effects). Examples of pharmaceuticallyacceptable salts include, but are not limited to, salts formed withcations (e.g., sodium, potassium, calcium, or polyamines such asspermine); acid addition salts formed with inorganic acids (e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, ornitric acid); and salts formed with organic acids (e.g., acetic acid,citric acid, oxalic acid, palmitic acid, or fumaric acid).

Pharmaceutical compositions containing the polypeptides of the presentinvention also can incorporate penetration enhancers that promote theefficient delivery of polypeptides to the skin of animals. Penetrationenhancers can enhance the diffusion of both lipophilic andnon-lipophilic drugs across cell membranes. Penetration enhancers can beclassified as belonging to one of five broad categories, i.e.,surfactants (e.g., sodium lauryl sulfate, polyoxyethylene-9-lauryl etherand polyoxyethylene-20-cetyl ether); fatty acids (e.g., oleic acid,lauric acid, myristic acid, palmitic acid, and stearic acid); bile salts(e.g., cholic acid, dehydrocholic acid, and deoxycholic acid); chelatingagents (e.g., disodium ethylenediaminetetraacetate, citric acid, andsalicylates); and non-chelating non-surfactants (e.g., unsaturatedcyclic ureas). Alternatively, inhibitory polypeptides can be deliveredvia iontophoresis, which involves a transdermal patch with an electricalcharge to “drive” the polypeptide through the dermis.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more polypeptides and (b) one or more other agentsthat function by a different mechanism. For example, anti-inflammatorydrugs, including but not limited to nonsteroidal anti-inflammatory drugsand corticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, can be included incompositions of the invention. Other non-polypeptide agents (e.g.,chemotherapeutic agents) also are within the scope of this invention.Such combined compounds can be used together or sequentially.

Compositions of the present invention additionally can contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, the compositions also can include compatible, pharmaceuticallyactive materials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or additional materials usefulin physically formulating various dosage forms of the compositions ofthe present invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers.Furthermore, the composition can be mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavorings,and aromatic substances. When added, however, such materials should notunduly interfere with the biological activities of the polypeptidecomponents within the compositions of the present invention. Theformulations can be sterilized if desired.

The pharmaceutical formulations of the present invention, which can bepresented conveniently in unit dosage form, can be prepared according toconventional techniques well known in the pharmaceutical industry. Suchtechniques include the step of bringing into association the activeingredients (e.g., the C_(H)2-C_(H)3 binding polypeptides of theinvention) with the desired pharmaceutical carrier(s) or excipient(s).Typically, the formulations can be prepared by uniformly and bringingthe active ingredients into intimate association with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product. Formulations can be sterilized if desired, provided thatthe method of sterilization does not interfere with the effectiveness ofthe polypeptide contained in the formulation.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention also can be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsfurther can contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol, and/or dextran. Suspensions also can contain stabilizers.

C_(H)2-C_(H)3 binding polypeptides of the invention can be combined withpackaging material and sold as kits for reducing Fc-mediated immunecomplex formation. Components and methods for producing articles ofmanufacture are well known. The articles of manufacture may combine oneor more of the polypeptides and compounds set out in the above sections.In addition, the article of manufacture further may include, forexample, buffers or other control reagents for reducing or monitoringreduced immune complex formation. Instructions describing how thepolypeptides are effective for reducing Fc-mediated immune complexformation can be included in such kits.

Methods for Using C_(H)2-C_(H)3 Binding Polypeptides to InhibitFc-Mediated Immune Complex Formation

C_(H)2-C_(H)3 binding polypeptides can be used in in vitro assays ofFc-mediated immune complex formation. Such methods are useful to, forexample, evaluate the ability of a C_(H)2-C_(H)3 cleft-bindingpolypeptide to block Fc-mediated immune complex formation. In vitromethods can involve, for example, contacting an immunoglobulin molecule(e.g., an antigen bound immunoglobulin molecule) with an effectormolecule (e.g., RF, FcR, FcRn, a histone, MBP, or another antibody) inthe presence and absence of a polypeptide of the invention, anddetermining the level of immune complex formation in each sample. Levelsof immune complex formation can be evaluated by, for example,polyacrylamide gel electrophoresis with Coomassie blue or silverstaining, or by co-immunoprecipitation. Such methods are known to thoseof ordinary skill in the art.

Methods provided herein also can be used to inhibit immune complexformation in a subject, and to treat an autoimmune disease in a subjectby inhibiting Fc-mediated immune complex formation in. Such methods caninvolve, for example, administering any of the polypeptides providedherein, or a composition containing any of the polypeptides providedherein, to a subject. For example, a method can include administering toan individual a composition containing a polypeptide that includes theamino acid sequence Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQID NO:10). Alternatively, a method can include administering to asubject a polypeptide that contains the amino acid sequenceAsp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:2), orAla-Pro-Pro-Asp-Cys-Ala-Arg-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ IDNO:14).

Methods provided herein can be used to treat a subject having, forexample, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE),lupus nephritis, autoimmune glomerulonephritis, atherosclerosis,multiple sclerosis (MS), Parkinson's disease, Crohn's disease,psoriasis, ankylosing spondylitis (AS), or cancer, or to a transplantrecipient. These conditions and the involvement of Fc-mediated immunecomplex formation are described in the subsections below. Methods of theinvention also can include steps for identifying a subject in need ofsuch treatment and/or monitoring treated individuals for a reduction insymptoms or levels of immune complex formation.

Rheumatoid Arthritis—RA is characterized by chronic joint inflammationthat eventually leads to irreversible cartilage destruction. In RA,abnormal IgG antibodies are produced by lymphocytes in the synovialmembranes. These abnormal IgG antibodies then act as antigens. Other IgGand IgM antibodies, termed Rheumatoid Factors (RF), are present in seraand synovia and subsequently react with these abnormal IgGantibody/antigens to produce immune complexes. Immune complexescontaining RF are abundant in synovial tissue of patients with RA. RFare directed to the Fc region of IgG, and interact with theC_(H)2-C_(H)3 cleft (Zack et al. (1995) J. Immunol. 155:5057-5063). Thepresence of RF is associated with systemic symptoms, joint erosion, andpoor prognosis, although the exact role of RF in RA remains to be fullyelucidated.

Collagen II (CII) induced arthritis, a murine model of RA, ischaracterized by polyarthritis, synovial hyperplasia, infiltration ofmononuclear cells, pannus formation and the destruction of cartilage andbone. Mice that are deficient for FcγRI and FcγRIII are protected fromCII induced arthritis, suggesting that blockade of FcγRs is useful fortreating RA (Kleinau et al. (2000) J. Exp. Med. 191:1611-1616). Whilethe etiology of RA is not fully understood, individuals that aregenetically predisposed to developing the disease produce high levels ofanti-CII antibodies. Immunization with immune complexes containing CIIproduces anti-idiotypic anti-CII antibodies that have been shown toactually be RF (Holmdahl et al. (1986) Scand. J. Immunol. 24:197-203).Inhibitors that bind to the IgG C_(H)2-C_(H)3 cleft will block thiscyclic production of anti-CII and RF anti-idiotypic antibodies.

The inflammation and subsequent cartilage damage caused by immunecomplexes in RA may be related to the occurrence of FcγRs on macrophages(Blom et al. (2000) Arthritis Res. 2:489-503). The absence of functionalFcγRI and FcγRIII in knock-out mice prevented inflammation and cartilagedestruction after induction of immune complex-mediated arthritis,whereas high basal expression of FcγRs on resident joint macrophages ofsimilarly treated mice susceptible to autoimmune arthritis wascorrelated with markedly more synovial inflammation and cartilagedestruction. In recent studies, the importance of these receptors ininflammation and tissue damage has been shown in various inflammatorydiseases, including autoimmune hemolytic anemia and thrombocytopenia,autoimmune glomerulonephritis, and induced glomerulonephritis. Since themajority of human RF bind to the IgG Fc C_(H)2-C_(H)3 cleft (Sasso etal. (1988) J. Immunol. 140:3098-3107; Corper et al. supra; and Sohi etal. supra), polypeptides that bind to the C_(H)2-C_(H)3 cleft woulddirectly inhibit the binding of RF to immune-complexed IgG Fc, andtherefore would ameliorate the contribution of RF to the pathology ofRA.

Systemic lupus erythematosus and lupus nephritis—SLE is a chronicautoimmune disease with many manifestations. The production ofautoantibodies leads to immune complex formation and subsequentdeposition in many tissues (e.g., glomeruli, skin, lungs, synovium, andmesothelium), leading to the manifestations of the disease. Renaldisease is common with SLE because the immune complexes often aredeposited in the renal glomeruli. Despite therapy, progression tochronic renal failure is common.

Lupus nephritis is an inflammation of the kidney that is caused bySLE-related glomerular deposition of immune complexes and FcγR (see,e.g., Clynes et al. (1998) Science 279:1052-1054). In mouse models ofSLE, significant proteinuria also was observed concomitant with theserological appearance of antibodies to DNA and histones, as well asimmune complexes of the IgG1, IgG2a, and IgG2b subclasses. The mediansurvival is 6 months, and mortality results from renal failure. B cellsand autoantibodies are thought to play essential roles in diseasedevelopment, and agents that interfere with autoantibody production havebeen shown to attenuate the disease.

Studies of the role of the FcγRs have been facilitated by theavailability of defined murine strains deficient in components of thispathway. The mouse strain γ^(−/−), which is deficient in the FcR γchain, does not express the activation receptors FcγRI and FcγRIII, butstill bears the inhibitory receptor FcγRIIB. Mice lacking FcγRI orFcγRIII were protected from developing Lupus nephritis. Through agenetic disruption of the FcγR/immune complex interaction, Clynes et al.(supra) showed that the interaction of immune complex and cellular Fcreceptors was essential to the development of Lupus nephritis. However,the mice lacking FcR still demonstrated significant renal immune complexdeposition.

Histone H1 has been shown to bind to immune complexes (Gussin et al.(2000) Ann. Rheum. Dis. 59:351-358; and Costa et al. (1984) J. Immunol.Methods 74:283-291). Costa et al. also showed that rheumatoid factorscompetitively inhibited the binding of histone H1 to immune complexes,suggesting that the binding of histone H1 to immune complexes involvesthe IgG Fc C_(H)2-C_(H)3 cleft. Other studies showed that perfusion ofrat kidneys with histones, DNA, and anti-DNA antibodies resulted in thedeposition of DNA/anti-DNA immune complexes in the glomerular basementmembrane (GBM), suggesting that immune complex binding to the GBM isdirectly mediated by the binding of histones to the GBM (Termaat et al.(1992) Kidney Int. 42:1363-1371; and Gussin et al. supra). The use ofpolypeptides that bind to the C_(H)2-C_(H)3 cleft would inhibit thebinding of histones to immune-complexed IgG Fc, and therefore wouldameliorate the contribution of these Fc-mediated immune complexes to thepathology of SLE and Lupus nephritis.

In a competitive inhibition study using IgG Fc fragments, both depositedIgG immune complexes and injected Fc fragments colocalized in themesangium of Fc-treated nephritic animals, suggesting that the blockadeof FcR could be the underlying mechanism of the beneficial effect of Fcfragments (Gómez-Guerrero et al. (2000) J. Immunol. 164:2092-2101). Thisstudy also demonstrated the central importance of immune complex to FcRinteractions in mediating Lupus nephritis. In addition, the reduction ofmultiple inflammatory cytokines demonstrated the importance ofpreventing the inflammatory cascade rather than attempting to interferewith the cascade by inhibiting one or more inflammatory molecules.Polypeptides that bind to the C_(H)2-C_(H)3 cleft therefore also wouldinhibit the binding of FcR to immune-complexed IgG Fc, and would reducethe contribution of FcR to the pathology of SLE and Lupus nephritis.

Gómez-Guerrero et al. also demonstrated that the elevated cholesterolobserved in untreated nephritis mice (227±27 mg/dl) was reduced by morethan half in nephritis mice treated with Fc fragments (103±16 mg/dl).Women between 35 and 44 years of age with systemic lupus erythematosushave a fifty times greater chance of developing advancedatherosclerosis/myocardial infarction than women of similar age withoutimmune complex disease (Manzi et al. (2000) Ann. Rheum. Dis.59:321-325). Although less dramatic, the same relationship holds truefor patients with rheumatoid arthritis. The accelerated rate ofatherosclerosis and myocardial infarction may be due to a chronicinflammatory state created by the formation of chronic immune complexes.The formation of these immune complexes can be prevented by inhibitorypolypeptides that bind to the IgG Fc C_(H)2-C_(H)3 cleft.

Autoimmune glomerulonephritis—Autoimmune glomerulonephritis, a disorderrelated to lupus nephritis, is due to a T cell dependent polyclonal Bcell activation that is responsible for production of antibodies againstself components (e.g., GBM, immunoglobulins, DNA, myeloperoxydase) andnon self components (e.g., sheep red blood cells and trinitrophenol).Increased serum IgE concentration is the hallmark of this disease.

Atherosclerosis—Atherosclerotic lesions are thought to be largely of aninflammatory nature. Recent studies have focused on the inflammatorycomponent of atherosclerosis, attempting to highlight the differencesbetween stable and unstable coronary plaques. An increasing body ofevidence supports the hypothesis that atherosclerosis shares manysimilarities with other inflammatory/autoimmune diseases. Indeed, thereare surprising similarities in the inflammatory/immunologic responseobserved in atherosclerosis, unstable angina, and rheumatoid arthritis,the prototype of autoimmune disease (Pasceri and Yeh (1999) Circulation100(21):2124-2126).

Activated macrophages and macrophage-derived foam cells laden withcholesterol esters are a major constituent of atherosclerotic lesions,and can influence lesion formation via several potential mechanisms. Onesuch mechanism is FcγR activation and/or FcγR-mediated clearance ofimmune complexes containing cholesterol, such as lipoprotein immunecomplexes. Recent studies indicated that highly cellular,preatheromatous lesions contain numerous macrophages in the zone ofproliferation that express each class of FcγR (FcγRIA, FcγRIIA, andFcγRIIIA; (Ratcliffe et al. (2001) Immunol. Lett. 77:169-174). Thesedata provided further support for the idea that FcγR-mediated clearanceof immune complexes can occur in arterial lesions during atherogenesis.Expression of both the high affinity (FcγRIA) and lower affinity(FcγRIIA/FcγRIIIA) receptors indicated that mono- and multivalentIgG-containing immune complexes could engage FcγR and influence lesionformation through several different inflammatory mechanisms triggered byreceptor activation.

There also appears to be an established link between chronic Chlamydiapneumoniae infections and atherosclerosis (Glader et al. (2000) Eur.Heart J. 21(8):639-646). The proatherogenic effects of C. pneumoniaelipoprotein may be enhanced and/or partly mediated through the formationof circulating immune complexes containing C. pneumoniae-specific IgGantibodies. The connection between chronic C. pneumoniae infections andatherosclerosis may be explained at least in part by an interaction withC. pneumoniae lipoprotein through the formation of circulating immunecomplexes. The C_(H)2-C_(H)3 binding polypeptides of the inventiontherefore also can be useful for treating elevated cholesterol levelsand atherosclerosis/myocardial infarction.

Multiple sclerosis—MS is an autoimmune disease that attacks theinsulating myelin sheath that surrounds neurons. This compromisesconduction of nerve signals between the body and brain. Symptoms can bemild or severe, short or long in duration, and may include blurredvision, blindness, dizziness, numbness, muscle weakness, lack ofcoordination and balance, speech impediments, fatigue, tremors, sexualdysfunction, and bowel and bladder problems. Although many people havepartial or complete remissions, symptoms for some progressively worsenwith few or no remissions.

Research has suggested that patients with MS have ongoing systemic virusproduction with resultant immune complex formation. In addition, MSpatients often have serum complexes containing brain-reactive components(Coyle and Procyk-Dougherty (1984) Ann. Neurol. 16:660-667). Theetiology of MS may be multifactorial and involve abnormal immunologicalresponses, possibly precipitated by infectious agents acquired duringchildhood by genetically susceptible individuals. The immunologicalresponses include alterations in myelin basic protein concentration,antimyelin antibody and immune complex activities in CSF, and in vitrostimulation, suppression, and migration inhibition of blood lymphocytes.These responses appear to correlate with stage of MS and severity of CNSdamage (Iivanainen (1981) J. Neuroimmunol. 1:141-172). Furthermore,levels of circulating immune complexes were found to be significantlyincreased in the sera of patients with progressive and activerelapsing-remittent MS (Procaccia et al. (1988) Acta Neurol. Scand.77:373-381). Immune complex levels also were found to be increased inthe cerebrospinal fluid of MS patients at the relapsing-remittent stage.

Myelin basic protein (MBP) is important in the immunopathogenesis of MS.MBP has been shown to bind to immune complexes and immune-complexed IgGFc (Sindic et al. supra). These immune complex binding sites were shownto be multivalent on MBP, and histones completely inhibited theagglutination of immune complexed IgG Fc latex-coated beads by MBP. Inaddition, certain FcR alleles have been correlated with the diseasecourse of MS (Vedeler et al. (2001) J. Neuroimmunol. 118:187-193). Theinvolvement of FcRs in MS was further suggested by studies showing thatFcRγ^(−/−) mice were protected from experimental autoimmuneencephalomyelitis, a model of MS induced by myelin oligodendrocyteglycoprotein (Abdul-Majid et al. (2002) Scand. J. Immunol. 55:70-81).Treating an MS patient with polypeptides that bind to the C_(H)2-C_(H)3cleft would inhibit the binding of MBP to immune-complexed IgG Fc andwould interfere with immune complex binding to FcRs, thereforeameliorating the pathology of MS.

Parkinson's disease—The clinical symptoms of Parkinson's disease (PD)result from the death of dopaminergic neurons in a section of the brainknown as the substantia nigra (SN). An overresponsive immune system mayplay a role in perpetuating PD by producing cytokines (e.g.,interleukin-1 and tumor necrosis factor) in response to the initialdamage, which can further injure cells in the brain. Furthermore,immunoglobulins from PD individuals have been shown to contribute to thepathogenesis of SN cells (Chen et al. (1998) Arch. Neurol.55:1075-1080).

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in thebiosynthesis of catecholamine neurotransmitters and is expressed only inthose neurons (e.g., the neurons of the SN) that normally synthesize andrelease such neurotransmitters. A structural analysis of TH suggeststhat immune complexes may bind to the enzyme and contribute to PDpathology. C_(H)2-C_(H)3 cleft-binding polypeptides therefore may beuseful for treating PD by inhibiting Fc-mediated binding of immunecomplexes to TH.

Crohn's disease—Crohn's disease results in chronic inflammation of thegastrointestinal tract, usually the small intestine. It affects about500,000 people in the United States, most often before age 30, causingmild to severe abdominal pain, diarrhea, fever and weight loss. Whilethe cause of the disease is unknown, the prevailing theory is that inCrohn's patients, the intestinal immune system over-reacts to viral orbacterial agents and initiates ongoing, uncontrolled inflammation of theintestine. It has been suggested that immune complexes of the IgG classmay activated inflammatory neutrophils in Crohn's disease (Nielsen etal. (1986) Clin. Exp. Immunol. 65:465-471).

RF and circulating immune complexes have been detected in the sera ofCrohn's patients (Procaccia et al. (1990) Boll Ist Sieroter Milan69:413-421; and Elmgreen et al. (1985) Acta Med. Scand. 218:73-78). Theprevalence of IgG-containing immune complexes and increased IgG RFlevels in these patients suggests that the inhibition of Fc-mediatedimmune complex formation would be useful for treating Crohn's disease.

Psoriasis—The release of cytokines such as interleukin-2 is thought tobe involved in psoriasis. In this disease, cytokines signal skin cellsto reproduce and mature at an accelerated rate, setting off otherreactions such as the activation of additional T cells and the“recruiting” of T cells into the skin. The initial activation of T cellsstarts a cycle that eventually leads to the formation of psoriasislesions on the surface of the skin.

The psoriasis-associated antigen, pso p27, is a major antigen in theimmune reactions of psoriasis. The synthesis of this particular antigenis reduced with the remission of inflammation in psoriatic skin lesions.See Dalaker et al. (1999) Acta Derm. Venereol. 79:281-284. Rabbitantisera against pso p27 antigen from psoriatic scale reacted with theFc region of human IgG. In addition, a commercial antiserum againsthuman IgG recognized a component in the pso p27-containing solution usedas the source of antigen for immunization of the rabbits (Asbakk et al.(1991) APMIS 99:551-556). The pso p27 antigen therefore may elicit theproduction of antibodies with rheumatoid factor activity in psoriaticpatients.

Anti-IgG activity at the cellular level in psoriasis patients has beendemonstrated using the so-called “rheumatoid” rosette test. The use ofpurified cell populations showed that the lymphocytes participating inthe rheumatoid rosette phenomenon were lacking conventional T and B cellmembrane markers. Such mononuclear cells bearing an FcR were able to actas killer cells to IgG-coated target cells. This cytotoxicity couldcontribute to the etiology of lesions in psoriasis (Clot et al. (1978)Brit. J. Derm. 99:25-30). Inhibiting the binding of such lymphocytes toIgG molecules with a C_(H)2-C_(H)3 binding polypeptide therefore wouldbe useful for treating psoriasis.

Ankylosing Spondylitis—Analysis of serum and synovial fluid samples frompatients with ankylosing spondylitis (AS) and from healthy blood donorsfor the presence of antibodies cross-reacting with the Fc region ofrabbit IgG revealed insignificant amounts of free RF, while IgG RF wereobserved in alkaline dissociated circulating immune complexes (CIC).Extensive amounts of IgG and moderate amounts of IgM reacting with psop27 also were detected in alkaline dissociated CIC from the AS patients(Rodahl et al. (1988) Ann. Rheum. Dis. 47:628-633). Antigens related topso p27 therefore appear to participate in CIC formation in AS, and maybe responsible for the elicitation of RF in patients with AS.

Cancer—Scientific evidence indicates that factors which can bind toimmunoglobulins can inhibit cancer metastasis (see, e.g., Mathiot et al.(1992) Immunol. Res. 11:296-304; and Hoover et al. (1990) Curr. Top.Mircobiol. Immunol. 166:77-85). Several key elements of the metastaticprocess can be inhibited by polypeptides and other compounds provided bythe invention. Fc receptors on cancer cells have been implicated incancer metastasis (see, e.g., Gergely et al. (1994) Adv. Cancer Res.64:211; Wallace et al. (1994) J. Leuk. Biol. 55:816-823; and Witz andRan. (1992) Immunol. Res. 11:283-295).

FcR positive tumor cells can bind to the Fc region of tumor-specificantibodies. FcRs thus can protect tumor cells by counteractingantibody-dependent effector functions such as complement-mediated lysisor antibody-dependent cell-mediated cytotoxicity (Gergely et al. supra).In this manner, FcR expression endows tumor cells with the ability toescape immune mechanisms. The expression of FcRs on tumor cells also mayfacilitate growth of the cells. In addition, tumor cells may use FcRs tobind to adhesion molecules and cause localized inflammatory responsesthat lead to angiogenesis. Tumor cells transfected in vitro with FcγRshowed higher rates of metastasis and tumorigenicity in vivo than cellsthat did not express the receptor (Witz and Ran supra). Use of aC_(H)2-C_(H)3 binding polypeptide to block interactions betweenimmunoglobulin molecules and FcRs on cancer cells would be useful forpreventing or reducing cancer metastasis.

Graft rejection following transplantation—C_(H)2-C_(H)3 bindingpolypeptides of the invention also are useful for preventing graftrejection following tissue or organ transplantation. Graft rejectiontypically results from the cumulative effects of both cell-mediated andhumoral immune attacks on the grafted tissue. Solid organ (tissue)transplantation includes, for example, transfer of kidney, heart, lungs,liver, pancreas, skin, cornea, and bone. Bone marrow transplantation isemployed in the treatment of conditions such as immunodeficiencydisease, aplastic anemia, leukemia, lymphoma, and genetic disorders ofhematopoiesis. Recent studies have suggested that FcR non-bindinganti-CD3 monoclonal antibodies profoundly affect T cell function bydelivering incomplete signals to activated T cells. These incompletesignals may result in functional inactivation of the inflammatory Th1 Tcell subset that mediates graft rejection. C_(H)2-C_(H)3 bindingpolypeptides of the invention also maybe useful for blocking signals toactivated T cells, thus inhibiting graft rejection.

The invention will be further described in the following examples, whichdoes not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Modeling the Amino Acid Residues within theC_(H)2-C_(H)3 Cleft That are Important for Binding to a Test Polypeptide

The first step in structure-based molecular drug design is determiningthe three-dimensional structure of the target receptor. Computerprograms (e.g., RasMol 2.6, Protein Explorer, or Chime, each availablefrom the University of Massachusetts Molecular Visualization web site onthe internet) that display the three-dimensional structure of a testligand, together with programs (e.g., Auto-dock or Dock) that displaythe exact three-dimensional structure of the target receptor, can beused to predict the structure of ligands that will bind to the targetreceptor. Three-dimensional structures can be produced by providing dataconsisting of the atomic coordinates of the target receptor and the testligand to a computer that contains the appropriate software. FIGS. 1Aand 1B show computer-generated, three-dimensional structures of an FcC_(H)2-C_(H)3 cleft from an IgG molecule in both a non-complexed and anantigen-bound state, revealing the open and closed conformationsdescribed herein. The atomic coordinates of a C_(H)2-C_(H)3 cleft fromIgG molecules complexed to a peptide ligand and a rheumatoid factor areshown in FIGS. 2A and 2B, respectively.

A computer-modeled examination of the interaction between an IgG FcC_(H)2-C_(H)3 cleft and a polypeptide with the amino acid sequenceAsp-Cys-Ala-Ala-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:39)showed that the Trp residue of the polypeptide could form a hydrogenbond to the cleft in between Ile253 of the C_(H)2 region and His435 ofthe C_(H)3 region (FIG. 3). The amino acids within the C_(H)2-C_(H)3cleft that were critical for binding to this polypeptide were Leu251,Met252, Ile253, Ser254, His433, Asn434, His435, and Tyr436.

Example 2 Amino Acid Substitutions within the Polypeptide of Example 1

Examination of a polypeptide having the amino acid sequenceArg-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:6) showedthat substitution of Arg for Asp in the first position did not affectbinding to the IgG Fc region. Replacement of the Trp in the fourthposition with Arg also is not expected to have a major impact onimmunoglobulin binding. The substitution of one or both of theseresidues to Arg (e.g., as set forth in SEQ ID NO:5) is expected to makethe polypeptide more water soluble, thereby increasing itsbioavailability. A three-dimensional structure of this modified peptidebound to the Fc C_(H)2-C_(H)3 region is shown in FIG. 3.

Example 3

In vitro assays for measuring ligand binding to the C_(H)2-C_(H)3 cleft.In vitro assays involving enzyme-linked immunosorbent assay (ELISA) anddouble immunodiffusion techniques are used to demonstrate competitiveinhibition of immune complexed IgG Fc binding to factors such as FcR,RF, FcRn, Clq, histones, CII, and MBP by polypeptides and compounds ofthe invention. Standardized reagents and ELISA kits are useful to reducecosts and increase the reproducibility of the experiments.

In a standard ELISA, an antigen is immunoadsorbed onto a plasticmicrowell. After suitable blocking and washing, a primary antibody withspecificity directed toward the antigen is added to the microwell. Afteranother wash phase, a secondary antibody that is directed toward theprimary antibody and conjugated to an enzyme marker such as horseradishperoxidase (HRP) is added to the microwell. Following another washcycle, the appropriate enzyme substrate is added. If an antigen toprimary antibody to secondary antibody/HRP conjugate is formed, theconjugated enzyme catalyzes a colorimetric chemical reaction with thesubstrate, which is read with a microplate reader or spectrophotometer.By standardizing the levels of the antigen and secondary antibody/HRPconjugate, a titer of the primary antibody (the variable) isestablished. In a standard ELISA system, the primary antibody binds tothe antigen through its complementarity determining regions (CDR)located in the Fab arms. Likewise, the secondary antibody/HRP conjugatebinds to the primary antibody via its CDR Fab region. Because the HRP isconjugated to the Fc region of the secondary antibody, direct Fc bindingis very limited or abrogated.

For this reason, a “reverse ELISA” technique is used to assess bindingof the Fc region to ligands that bind to immune complexed IgG Fc. In areverse ELISA, the enzyme (e.g., HRP) is not covalently conjugated tothe Fc portion of the secondary antibody. Rather, a preformed immunecomplex of peroxidase-rabbit (or mouse) anti-peroxidase IgG (“PAP”complex) is used. In this method, HRP serves as the enzyme marker butdoes not block the Fc region. In the reverse ELISA system, an FcC_(H)2-C_(H)3 cleft binding ligand (e.g., purified human Clq) is boundto microwell plates. In the absence of competitor, PAP complexes bind tothe immobilized ligand and the reaction between HRP and its substrateproduces a signal. This signal is reduced by polypeptides and compoundsof the invention that inhibit PAP binding to the immobilized ligand.

Inhibition of Clq binding: Twenty μl of peroxidase (P) (Sigma) wasdiluted in 2 ml of sample diluent (Quidel Corp, San Diego, Calif.).Twenty μl of anti-peroxidase (AP) (Sigma) was diluted in 2 ml of samplediluent, and 20 μl of the diluted AP was added to the diluted P (1:100antigen:antibody ratio) to form peroxidase-anti-peroxidase (PAP)complexes. The extreme antigen excess guaranteed that any singleantibody would be bound to two peroxidase molecules and larger immunecomplexes would not form, thus preventing the bridging of larger immunecomplexes to multimeric Clq (heximeric). One hundred μl of peptide orClq was pre-incubated with freshly prepared PAP for 30 minutes, added toClq coated plates (Quidel Corp.), and incubated for one hour. Afterwashing, ABTS substrate (Quidel Corp.) was added to the plates andincubated for 30 minutes, and the plates were read at 405 nm. Allpeptides were cyclized by forming a disulfide bond between the twocysteine residues. Results are shown in Table 1.

Soluble Clq resulted in the lower OD 405 value, and thus provided thegreatest competitive inhibition of solid phase bound Clq to immunecomplexes. Most of the other peptides prevented binding of immunecomplexes to solid phase Clq, with APPCARHLGELVWCT (SEQ ID NO:14) givingthe next lowest OD value. The alanine-substituted peptide(DCAAHLGELAACT; SEQ ID NO:40), with key binding residues substituted toalanine, resulted in an OD reading that was not significantly differentfrom the positive control. TABLE 1 SEQ ID Peptide NO: OD 405 nm (+)Control (PAP with no peptide) — 0.467 DCAAHLGELAACT 40 0.458DCAWHLGELVWCT 2 0.208 APPCARHLGELVWCT 14 0.163 PCARHLGELVWCT 41 0.205RCARHLGELVWCT 5 0.247 DCARHLGELVWCT 4 0.193 C1q (control inhibitor) —0.149

Inhibition of FcR binding: Once the reverse ELISA protocol wasestablished using the Clq assay, the assay was redesigned using FcγIIa,FcγIIb and FcγIII in place of Clq. Highly purified FcγIIa, FcγIIb andFcγIII were immunoadsorbed onto plastic microwells. After optimizing theFcγR reverse ELISA system, simple competitive inhibition experimentsusing polypeptides of the invention were conducted to investigate theirability to inhibit binding of immune complexes to purified FcγR.

Falcon microtiter plates were coated with 1:10 dilutions of highlypurified FcγIIa, FcγIIb and FcγIII and incubated for 24 hours. Theplates were washed and then blocked with 5×BSA blocking solution (AlphaDiagnostic International, San Antonio, Tex.) for 24 hours. Equal amounts(50 μl) of peptide and 1:10 PAP immune complexes were pre-incubated forone hour and then incubated on the FcR coated plates for one hour. Afterwashing, plates were incubated with TMB substrate (Alpha DiagnosticInternational) for 30 minutes. Stop solution (10 μl) was added and theplates were read at 450 nm. Results are shown in Table 2.

Since large amounts of soluble FcR were not available; a positivecontrol inhibitor was not included for these experiments. Of thepeptides tested, DCAWHLGELVWCT (SEQ ID NO:2) caused the greatestinhibition of binding, while APPCARHLGELVWCT (SEQ ID NO:14) resulted inthe second lowest OD reading. TABLE 2 SEQ ID Peptide NO: FcγIIa FCγIIbFcγIII (+) Control — 3.600 3.600 3.600 (PAP with no peptide)DCAAHLGELAACT 40 2.434 2.263 2.413 DCAWHLGELAACT 42 1.484 1.067 1.345DCAWHLGELVWCT 2 0.499 0.494 0.477 APPCARHLGELVWCT 14 0.682 0.554 0.542PCARHLGELVWCT 41 1.149 1.211 1.602 RCARHLGELVWCT 5 3.398 3.284 3.502DCARHLGELVWCT 4 2.539 1.952 2.529

Inhibition of RF binding: The assay to test inhibition of RF binding tothe IgG Fc C_(H)2-C_(H)3 clefts was very similar to the Clq-CIC EIAassay described above, with the exception that polyclonal IgM RF wascoated onto the microwells instead of Clq. After optimization, the samecompetitive inhibition techniques as described for the Clq-CIC EIA wereused to demonstrate inhibition of polyclonal RF to immune complexbinding. High titer, RF positive sera were purchased from ResearchDiagnostics (Flanders, N.J.).

A 1:10 dilution of 200 μl of 200 I.U. rheumatoid factor (RF) (+) control(positive standard provided by Research Diagnostics was coated ontoFalcon microtiter plates and incubated for 24 hours. The plates wereblocked with 1:5 BSA blocking buffer (Alpha Diagnostic International)for one hour. Freshly prepared 1:10 PAP (antigen:antibody) immunecomplexes were pre-incubated for 30 minutes with peptides or RF(positive control containing only buffer). After washing, plates wereincubated with ABTS substrate (Research Diagnostics) for 30 minutes andthen read at 405 mn. Results are shown in Table 3. TABLE 3 SEQ IDPeptide NO: OD 405 nm (+) Control (PAP with no peptide) — 0.753DCAWHLGELAACT 42 0.622 DCAWHLGELVWCT 2 0.163 APPCARHLGELVWCT 14 0.103PCARHLGELVWCT 41 0.106 RCARHLGELVWCT 5 0.152 DCARHLGELVWCT 4 0.109 RF(control inhibitor) — 0.108

Soluble rheumatoid factor (RF) provided inhibition of solid phase RFbinding to immune complexes. Peptides APPCARHLGELVWCT (SEQ ID NO:14),PCARHLGELVWCT (SEQ ID NO:41), and DCARHLGELVWCT (SEQ ID NO:4) had ODreadings essentially identical to that of soluble RF, and thus providedvery effective inhibition of RF binding to immune complexes.

Inhibition of histone binding: The binding of immune complexes to thekidneys in lupus nephritis appears to involve (a) the binding ofhistones to the GBM, and then (b) the binding of immune complexes(through the IgG Fc C_(H)2-C_(H)3 cleft) to the bound histones.Experiments similar to those described above were used to inhibitbinding of purified histone to IgG Fc binding. Histone (Sigma) wasdiluted 1:10 in coating buffer (Alpha Diagnostic International) andincubated on Falcon microtiter plates for 24 hours. Plates were blockedwith 5×BSA blocking solution (Alpha Diagnostic International) for 24hours. Freshly prepared 1:10 rabbit PAP (Sigma) was pre-incubated witheither peptides or histone for one hour, and 100 μl of the mixture wasadded to the histone-coated plates for one hour. Plates were incubatedwith ABTS substrate (Quidel Corp.) for 45 minutes, and OD 405 was read.Results are shown in Table 4.

The DCAWHLGELVWCT peptide (SEQ ID NO:2) was best peptide inhibitor, withthe second lowest OD value. TABLE 4 Peptide SEQ ID NO: OD 405 nm (+)control (buffer only) — 1.268 DCAWHLGELAACT 42 0.729 DCAWHLGELVWCT 20.212 APPCARHLGELVWCT 14 0.368 PCARHLGELVWCT 41 0.444 RCARHLGELVWCT 50.359 DCARHLGELVWCT 4 0.363 Histone — 0.057

Experiments also were conducted to test the ability of a polypeptidehaving the sequence set forth in SEQ ID NO:16 (also referred to asNB-406) to inhibit the binding of histones to IgG PAP immune complexes.The results are shown in Table 5. This peptide was able to significantlyinhibit the binding of histones to IgG PAP. TABLE 5 Peptide SEQ ID NO:OD 405 nm APPDCAWHLGELVWCT 16 0.227 Positive Control — 2.110

Inhibition of MBP binding: MBP (Sigma) was diluted 1:10 with coatingbuffer (Alpha Diagnostic International) and incubated for 24 hours onFalcon microtiter plates. Plates were washed and then blocked with 5×BSAblocking buffer (Alpha Diagnostic International) for 24 hours. Rabbit1:10 PAP immune complexes were pre-incubated with equal amounts ofpeptide or MBP for 30 minutes. One hundred μl of PAP immunecomplexes/peptide or PAP/MBP was then added to the MBP-coated plates andincubated for one hour. The plates were washed and incubated with TMBsubstrate (Alpha Diagnostic International) for 30 minutes. After addingstop solution (Alpha Diagnostic International), the plates were read at450 nm. Results are shown in Table 6.

With the exception of the peptide substituted with alanine at positions10 and 11 (SEQ ID NO:42), the peptides tested showed a varying amount ofinhibition of solid phase MBP binding to immune complexes. TABLE 6Peptide SEQ ID NO: OD 405 nm MBP — 0.139 DCAWHLGELAACT 42 0.706DCAWHLGELVWCT 2 0.588 APPCARHLGELVWCT 14 0.466 PCARHLGELVWCT 41 0.489RCARHLGELVWCT 5 0.569 DCARHLGELVWCT 4 0.473 (+) Control (buffer only) —1.033

Further experiments were conducted to determine the effect of SEQ IDNO:16 (also referred to as NB-406) on binding of MBP to IgG PAP immunecomplexes. The results are shown in Table 7; SEQ ID NO:16 was able toinhibit the interaction between IgG PAP immune complexes and MPB. TABLE7 Peptide SEQ ID NO: OD 405 nm APPDCAWHLGELVWCT 16 0.500 PositiveControl — 2.343

Inhibition of Fc:Fc interactions: The Fc region of IgG4 interacts in anFc to Fc fashion with immune complexed IgG. Purified IgG4 is used withpolypeptides of the invention to examine inhibition of immune complexformation and Fc:Fc interactions. Chemical modification of His435, acritical IgG Fc amino acid bound by polypeptides of the invention, isknown to inhibit Fc:Fc interactions.

The assay to test the ability of polypeptides of the invention tointerfere with Fc:Fc binding is very similar to the Clq-CIC EIA assaydescribed above, with the exception that whole human IgG4 is coated ontothe microwells instead of Clq. After optimizing this CIC assay, the samecompetitive inhibition techniques as described for the Clq-CIC EIA areused to demonstrate inhibition of IgG4 Fc:Fc immune complex binding.Results are shown in Table 8.

Of the peptides tested, DCARHLGELVWCT (SEQ ID NO:4), DCAWHLGELVWCT (SEQID NO:2), and APPDCARHLGELVWCT (SEQ ID NO:14) provided the strongestinhibition of Fc:Fc binding. TABLE 8 Peptide SEQ ID NO: OD 405 nm IgG4 —0.108 DCAWHLGELAACT 42 0.557 DCAWHLGELVWCT 2 0.107 APPDCARHLGELVWCT 140.107 PCARHLGELVWCT 41 0.129 RCARHLGELVWCT 5 0.191 DCARHLGELVWCT 4 0.096Positive Control (buffer) — 0.716

Inhibition of Collagen II and FcRn binding: Polypeptides of theinvention also are tested for their ability to inhibit binding of CII toanti-CII antibodies and binding of FcRn to immune complexes. The assayto test the ability of polypeptides of the invention to interfere withsuch binding is very similar to the Clq-CIC EIA assay described above,with the exception that the microwells are coated with CII or FcRninstead of Clq. The immunodominate CII peptide is a small linear peptideand is readily synthesized, and purified CII extracts also arecommercially available. After optimization, the same competitiveinhibition techniques as described for the Clq-CIC EIA are used todemonstrate inhibition of binding.

There are many similarities in the interactions of FcRn and thepolypeptides provided herein (e.g., SEQ ID NO:16) with the IgGC_(H)2-C_(H)3 cleft. For example, both FcRn and the polypeptidesprovided herein use hydrophobic packing/burial of hydrophobic residuesas the primary binding force. In particular, for example, bothpolypeptides use Trp residues as primary amino acid contacts—Trp 133(rat FcRn) or Trp 131 (human FcRn), as well as Trp 14 and Val 13 of SEQID NO:16 or other peptides provided herein bind to IgG by burial ofhydrophobic residues at or near Ile 253. Further, both FcRn and thepolypeptides provided herein bind more avidly in acidic pH due to thepKa of IgG His 435, which is in the pH 6-7 range. FcRn binds with highavidity at pH≦6.5 when His 435 is positively charged, and with lowavidity upon deprotonation of His-435 at pH values above 7.0 (Martin etal. (2001) Mol. Cell 7:867-877; West et al. (2000) Biochem.39:9698-9708; and DeLano et al. (2000) Science 287:1279). Thepolypeptides provided herein also show some pH dependence (aboutfour-fold) due to the pKa of His-435. FIGS. 5 and 6 show thethree-dimensional similarities of the binding of FcRn and SEQ ID NO:16to IgG Fc. Since the peptides provided herein can bind with high avidityover a wider pH range, they can be effective inhibitors of FcRn-IgG Fcbinding, as well as effective inhibitors of binding of other moleculesto IgG Fc.

Example 4 Inhibition of Rheumatoid Factor Binding to Monomeric IgG

The ability of the peptides to inhibit the binding of rheumatoid factorto monomeric IgG was tested. Binding to monomeric IgG may be important,as it may increase the half-life of particular peptides and allow themto be more bioactive.

A standard rheumatoid factor commercial test was used (ResearchDiagnostics) with the following modifications: 100 μl of test peptideswere pre-incubated for 30 minutes with human monomeric IgG (ResearchDiagnostics). Plates were washed and incubated with 200 I.U. rheumatoidfactor positive control supplied with the test kit. The rest of the testwas performed according to the manufacturer's instructions. Results areshown in Table 9.

A RF control was not used for this experiment. Of the peptides tested,DCAWHLGELVWCT (SEQ ID NO:2) clearly out-performed the others, giving thelowest OD reading. TABLE 9 Peptide SEQ ID NO: OD 405 nm (+) Control(buffer only) — 1.376 DCAAHLGELAACT 40 1.421 DCAWHLGELAACT 42 1.397DCAWHLGELVWCT 2 0.464 APPCARHLGELVWCT 14 1.393 PCARHLGELVWCT 41 1.323RCARHLGELVWCT 5 1.314 DCARHLGELVWCT 4 1.231

Example 5 Inhibition of RF Binding to Immune Complexes Using AdditionalPeptides

The ability of additional peptides to inhibit the binding of RF toimmune complexes was tested. Immune complexes (PAP) were formed bymixing 2 μl of rabbit anti-peroxidase with 50 μl of peroxidase in 1 mldistilled water. PAP (100 μl) were pre-incubated with 100 μl of peptidefor one hour. Plates coated with RF were blocked with 5×BSA for 24hours. The PAP/peptide mixtures (100 μl) were incubated with the RFcoated plates for 30 minutes. RF (100 μl of a 200 I.U. standard suppliedby Research Diagnostics) was used as a negative control. After washingand incubation with ABTS substrate (Quidel Corp., San Diego, Calif.) for15 minutes, plates were read at 405 nm.

Results are shown in Table 10. TABLE 10 Peptide SEQ ID NO: OD 405 nmDCAWHLGELVWCT 2 0.218 APPCARHLGELVWCT 14 0.358 DCAFHLGELVWCT 3 0.267APPDCAWHLGELVWCT 16 0.205 APPCAFHLGELVWCT 15 0.226 APPCAWHLGELVWCT 130.250 RF (negative control) — 0.104 Positive Control — 1.176

All peptides tested resulted in similar rates of inhibition, withAPPDCAWHLGELVWCT (SEQ ID NO:16) providing the best inhibition.

Example 6 Inhibition of Clq Binding to Immune Complexes Using AdditionalPeptides

PAP complexes were formed as described in Example 5, and 100 μl werepre-incubated with 100 μl of peptide or human Clq (Quidel Corp.) for onehour. The Clq/PAP and peptide/PAP mixtures (100 μl) were incubated withClq coated plates for 30 minutes. After washing, plates were incubatedwith ATBS (Quidel Corp.) for 15 minutes and read at 405 nm. Results areshown in Table 11.

As in Example 5, APPDCAWHLGELVWCT (SEQ ID NO:16) resulted in thegreatest inhibition of Clq binding, almost equaling Clq itself. PeptideAPPCARHLGELVWCT (SEQ ID NO:14) gave the next best result. TABLE 11Peptide SEQ ID NO: OD 405 nm DCAWHLGELVWCT 2 1.100 APPCARHLGELVWCT 140.567 DCAFHLGELVWCT 3 0.859 APPDCAWHLGELVWCT 16 0.389 APPCAFHLGELVWCT 150.983 APPCAWHLGELVWCT 13 1.148 C1q (negative control) — 0.337 PositiveControl — 2.355

Example 7 Inhibition of RF Binding to Monomeric IgG by AdditionalPeptides

The ability of additional peptides to inhibit RF binding to monomericIgG was tested. A standard rheumatoid factor commercial test was used(Research Diagnostics, New Jersey) with the following modifications: 100μl of test peptides were pre-incubated for 1 hour with human monomericIgG (Research Diagnostics, New Jersey). Plates were then washed andincubated with 200 I.U. of the RF positive control supplied with thetest kit. The rest of the test was performed according to themanufacturer's instructions. Results are shown in Table 12.

Peptide DCAWHLGELVWCT (SEQ ID NO:2) resulted in the greatest inhibitionof RF binding to monomeric IgG, followed by peptide DCAFHLGELVWCT (SEQID NO:3). TABLE 12 Peptide SEQ ID NO: OD 405 nm DCAWHLGELVWCT 2 0.539APPCARHLGELVWCT 14 1.095 DCAFHLGELVWCT 3 0.962 APPDCAWHLGELVWCT 16 1.065APPCAFHLGELVWCT 15 1.159 APPCAWHLGELVWCT 13 1.166 Positive Control —1.312

Example 8 Inhibition of FcR Binding to PAP by Additional Peptides

Falcon microtiter plates were coated with 1:10 dilutions of highlypurified FcγIIa, FcγIIb and FcγIII, sealed, and incubated for one yearat 4° C. The plates were washed and then blocked with 5×BSA blockingsolution (Alpha Diagnostic International, San Antonio, Tex.) for 24hours. PAP immune complexes were formed as described in Example 5. PAP(100 μl) were pre-incubated with 100 μl of peptide for one hour.PAP/peptide mixtures were added to the FcR coated plates and incubatedfor one hour. After washing, plates were incubated with ABTS substratefor 15 minutes and read at 405 nm. Results are shown in Table 13.

Peptide APPDCAWHLGELVWCT (SEQ ID NO:16) appeared to result in thegreatest inhibition of FcR binding to PAP, followed by peptideDCAWHLGELVWCT (SEQ ID NO:2). TABLE 13 Peptide SEQ ID NO: FcγIIa FcγIIbFcγIII DCAWHLGELVWCT 2 0.561 0.532 0.741 APPCARHLGELVWCT 14 0.956 0.7680.709 DCAFHLGELVWCT 3 0.660 0.510 0.810 APPDCAWHLGELVWCT 16 0.509 0.4960.670 APPCAFHLGELVWCT 15 0.605 0.380 0.880 APPCAWHLGELVWCT 13 0.6580.562 0.530 Positive Control — 1.599 1.394 1.588

Example 9 In Vivo Assay of FD5 Therapeutic Efficacy of in a Murine Modelof Collagen-Induced Arthritis

DBA/1J mice were obtained from Jackson Laboratories (Bar Harbor, Me.),and were maintained in quarantine with daily inspection for four days.Once the animals were determined to be in overt good health, they werereleased from quarantine to routine maintenance.

On day-2, 10 mg of collagen (Sigma Chemical Co., St. Louis, Mo.) wasdissolved in 5 ml 0.01 M acetic acid and stirred at 4-8° C. overnight.On day-1, adjuvant was prepared by suspending 10.6 mg Mycobacteriumtuberculosis (Difco) in 5.3 ml squalene (Sigma). The suspension washomogenized throughout the day.

On day 0, 3.7 ml of the adjuvant suspension was emulsified with 3.7 mlof the collagen solution. The mice were ear tagged for identificationpurposes, weighed, and anesthetized with isoflurane. Ninety “disease”mice were injected intradermally with 0.05 ml of the adjuvant/collagenemulsion. Ten control mice (“non-disease”) received an intradermalinjection of 0.01 M acetic acid/squalene. No adverse reactions to theinjection procedures were observed, and the animals were returned toroutine maintenance.

Fresh preparations of collagen and adjuvant were prepared on days 6 and13. On days 7 and 14, mice were anesthetized and injected as they hadbeen on day 0. Again, no adverse reactions to the injection procedurewere noted after either injection.

Mice were examined daily for symptoms of arthritis, beginning on day 15.The first symptoms (a single swollen digit) were observed in three miceon day 21. By day 31, 50 percent of the disease mice were symptomatic,while none of the non-disease mice were symptomatic. The degree ofarthritic symptoms was scored for each individual mouse as follows: 0,normal; 2.5, slight focal chronic erosive osteoarthritis; 5, moderatefocal suppurative erosive osteoarthritis; 10, moderate multifocalchronic erosive osteoarthritis. On day 32, disease mice were weighed,scored for arthritic symptoms, and divided into nine treatment groups often mice each. Each group had a similar average arthritic index. Bloodwas drawn from each mouse for standard chemistry/CBC analysis.

Polypeptides “ID 14” and “ID 2” having the amino acid sequences setforth in SEQ ID NOS:14 and 2, respectively, were obtained from SigmaGenosys (The Woodlands, Tex.). On day 32, 305.2 mg ID 14 was dissolvedin 91.7 ml phosphate buffered saline (PBS), pH 7.4, yielding a 3.33mg/ml solution. 248.7 mg ID 2 was dissolved in 74.7 ml PBS to yield a3.33 mg/ml solution. These were aliquotted and frozen at −20° C. forfuture use.

REMICADE® was obtained from Centocor (Malvern, Pa.). A 3.33 mg/mlsolution was prepared by dissolving 100 mg REMICADE® in 30.03 ml PBS.Aliquots of this solution were stored at −20° C. A solution ofprednisolone 21-hemisuccinate (Sigma) was prepared by dissolving 14.1 mgin 15 ml PBS. Aliquots were stored at ambient temperature.

Following the blood draw, the ninety disease mice were divided into ninegroups of ten mice each. The groups were injected subcutaneously withvehicle, 1 mg/kg ID 14, 10 mg/kg ID 14, 100 mg/kg ID 14, 1 mg/kg ID 2,10 mg/kg ID 2, 100 mg/kg ID 2, 3 mg/kg prednisolone, or 10 mg/kgREMICADE®, each at a volume of 30 ml/kg. Mice were weighed and scoredfor arthritic symptoms daily from day 33 through day 47. In addition,mice received daily injections of vehicle, ID 14, ID 2, prednisolone, orREMICADE® as on day 32. Injection sites were examined daily, and noadverse reactions were observed.

On day 48, the mice were weighed and scored for arthritic symptoms. Allanimals were anesthetized and exsanguinated for standard chemistry/CBCanalysis. Hindlimbs were removed and placed in 10% buffered fonnalin forhistological analysis, to examine the extent of inflammatory lesionsinvolving the synovial membranes, articular cartilage, periarticulartissues, and bone. The analysis of each hindlimb was graded using thefollowing scale: 0, normal; 2.5, slight focal chronic erosiveosteoarthritis; 5, moderate focal suppurative erosive osteoarthritis;10, moderate multifocal chronic erosive osteoarthritis. TABLE 14 In vivoArthritis Study Arthritic Percent Histological Percent Index ChangeEvaluation Change Diseased (control) 3.8 — 45 — ID 14 (1 mg/kg) 2.8 −26%55 +22% ID 14 (10 mg/kg) 2.7 −29% 40 −11% ID 14 (100 mg/kg) 3.6  −5% 45 0% ID 2 (1 mg/kg) 2.8 −26% 25 −44% ID 2 (10 mg/kg) 2.4 −37% 25 −44% ID2 (100 mg/kg) 2.5 −34% 10 −78% REMICADE ® (10 mg/kg) 3.8  0% 45  0%Prednisolone (3 mg/kg) 1.4 −63% 25 −44%

Calculation of average arthritic indices for the various groups over thefinal three days of treatment revealed that administration of 1 mg/kgand 10 mg/kg ID 14 resulted in a 26-29% reversal of arthritic symptoms(FIG. 4A and Table 14). The 100 mg/kg dose of ID 14 did not have asignificant effect on the disease. Daily injection of 1, 10, or 100mg/kg ID 2 resulted in a dose-dependent reversal of arthritic symptoms(FIG. 4B and Table 14). A maximum inhibition of 37% was observed withthe 10 mg/kg dose. By comparison, treatment with prednisolone preventedfurther development of arthritic symptoms, relative to vehicle-treateddisease rats (FIG. 4C and Table 14). A maximum reversal of 63% ofarthritic symptoms was observed after eight days of treatment withprednisolone. In contrast, treatment with REMICADE® had no effect onarthritic symptoms (FIG. 4C). Thus, polypeptides ID 2 and ID 14 wereable to reverse arthritic symptoms in these animals.

Histological examination of the hindlimbs from the mice revealed thatadministration of ID 2 at 1 mg/kg, 10 mg/kg, and 100 mg/kg resulted in a44-78% reversal of arthritic symptoms (Table 14). Daily injection of 1mg/kg, 10 mg/kg, and 100 mg/kg of ID 14 did not have a significanteffect. Treatment with prednisolone resulted in a 44% reversal ofarthritic symptoms. In contrast, treatment with REMICADE® had no effecton histological symptoms of arthritis. Thus, polypeptide ID 2 was ableto reverse the arthritic symptoms in these animals.

Example 10 In Vivo Assays for Assessing Inhibition of Fc-Mediated ImmuneComplex Formation in a Mouse Model of RA

The inhibitory effects of polypeptides of the invention also are testedin animal models of CII-induced arthritis. Arthritis prone DBA/1 miceare injected intradermally with 100 μg of bovine CII emulsified inComplete Fruends Adjuvant. These mice typically develop RA-like diseaseafter 60 days. Mice are divided into three groups: (1) a control groupthat is expected to develop arthritis; (2) a group treated withpolypeptides or compounds of the invention at the time of CIIimmunization; and (3) a group treated with polypeptides or compounds ofthe invention beginning 45-60 days after CII immunization, in mice thathave already started showing signs of arthritis. Symptoms of arthritisbefore and after treatment are monitored to determine the in vivoeffectiveness of polypeptides and compounds of the invention.

Example 11 In Vivo Assays for Assessing Inhibition of Fc-Mediated ImmuneComplex Formation in a Mouse Model of SLE

MRL/MpJ-Fas (MRL/lpr) mice develop a syndrome that is serologically andpathologically similar to human SLE. These mice have high levels of IgGautoantibodies to nuclear antigens such as single-stranded anddouble-stranded DNA, and also exhibit progressive glomerulonephritis asa result of in vivo immune complex formation and deposition in theglomerulus of the kidneys. At seven weeks of age, MRL/lpr mice aretreated with biweekly intraperitoneal injections of the polypeptidesdescribed herein. Levels of proteinuria are measured once weekly forforty weeks, to determine whether animals treated with the polypeptideshave lower levels of proteinuria. After forty weeks, renal biopsies areconducted to determine whether the treated animals have lessglomerulonephritis and/or IgG immune complex deposition. In addition,mean survival rates are calculated to determine if the mean survival ofthe treated animals is increased. Similar studies are conducted using(NZB×NZW)F1 mice, another murine model of SLE.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for inhibiting immune complex formation in a subject, saidmethod comprising administering to said subject a composition comprisinga purified polypeptide, said polypeptide comprising the amino acidsequence Cys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:8),wherein Xaa is Arg, Trp, Tyr, or Phe.
 2. The method of claim 1, whereinsaid immune complex formation is associated with rheumatoid arthritis.3. The method of claim 2, further comprising the step of monitoring saidsubject for clinical or molecular characteristics of rheumatoidarthritis.
 4. The method of claim 1, wherein said polypeptide furthercomprises a terminal stabilizing group.
 5. The method of claim 4,wherein said terminal stabilizing group is at the amino terminus of saidpolypeptide and is a tripeptide having the amino acid sequenceXaa-Pro-Pro, wherein Xaa is any amino acid.
 6. The method of claim 5,wherein Xaa is Ala.
 7. The method of claim 4, wherein said terminalstabilizing group is at the carboxy terminus of said polypeptide and isa tripeptide having the amino acid sequence Pro-Pro-Xaa, wherein Xaa isany amino acid.
 8. The method of claim 7, wherein Xaa is Ala.
 9. Themethod of claim 4, wherein said terminal stabilizing group is a smallstable protein.
 10. The method of claim 9, wherein said small stableprotein comprises a four-helix bundle topology.
 11. The method of claim9, wherein said small stable protein is Rop.
 12. The method of claim 1,wherein said polypeptide further comprises an additional amino acid atthe amino terminus of said amino acid sequence.
 13. The method of claim12, wherein said additional amino acid is any amino acid other than Cys.14. The method of claim 12, wherein said additional amino acid is Asp.15. The method of claim 1, wherein said polypeptide has a length ofabout 10 to about 50 amino acids.
 16. The method of claim 1, whereinsaid polypeptide comprises the amino acid sequenceAsp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:2). 17.The method of claim 1, wherein said polypeptide comprises the amino acidsequenceTrp-Glu-Ala-Asp-Cys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Lys-Val-Glu-Glu(SEQ ID NO:32).
 18. A method for treating rheumatoid arhritis, saidmethod comprising identifying an individual with rheumatoid arthritis orat risk for developing rheumatoid arthritis, and administering to saidindividual a composition comprising a purified polypeptide, saidpolypeptide comprising the amino acid sequenceCys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:8), whereinXaa is Arg, Trp, Tyr, or Phe.
 19. The method of claim 18, furthercomprising the step of monitoring said subject for clinical or molecularcharacteristics of rheumatoid arthritis.
 20. The method of claim 18,wherein said polypeptide further comprises an Asp at the amino terminusof said amino acid sequence.
 21. The method of claim 18, wherein saidpolypeptide further comprises a terminal stabilizing group.
 22. Themethod of claim 21, wherein said terminal stabilizing group is at theamino terminus of said polypeptide and is a tripeptide having the aminoacid sequence Xaa-Pro-Pro, wherein Xaa is any amino acid.
 23. The methodof claim 22, wherein Xaa is Ala.
 24. The method of claim 21, whereinsaid terminal stabilizing group is at the carboxy terminus of saidpolypeptide and is a tripeptide having the amino acid sequencePro-Pro-Xaa, wherein Xaa is any amino acid.
 25. The method of claim 24,wherein Xaa is Ala.
 26. The method of claim 21, wherein said terminalstabilizing group is a small stable protein.
 27. The method of claim 26,wherein said small stable peptide comprises a four-helix bundletopology.
 28. The method of claim 26, wherein said small stable proteinis Rop.
 29. The method of claim 18, wherein said polypeptide has alength of about 10 to about 50 amino acids.
 30. The method of claim 18,wherein said polypeptide comprises the amino acid sequenceAsp-Cys-Ala-Trp-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:2). 31.The method of claim 18, wherein said polypeptide comprises the aminoacid sequenceTrp-Glu-Ala-Asp-Cys-Ala-Xaa-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-Lys-Val-Glu-Glu(SEQ ID NO:32).
 32. A purified polypeptide comprising the amino acidsequence Xaa₁-Pro-Pro-Cys-Ala-Xaa₂-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr(SEQ ID NO:12), wherein Xaa₁ is any amino acid and Xaa₂ is Arg, Trp,Tyr, or Phe.
 33. The purified polypeptide of claim 32, wherein Xaa₁ isAla.
 34. A composition comprising the polypeptide of claim
 32. 35. Apurified polypeptide comprising the amino acid sequenceCys-Ala-Phe-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr (SEQ ID NO:9).
 36. Thepurified polypeptide of claim 35, wherein said polypeptide comprises nomore than about 20 amino acids.
 37. The purified polypeptide of claim35, wherein said polypeptide further comprises a terminal stabilizinggroup.
 38. The purified polypeptide of claim 37, wherein said terminalstabilizing group is at the amino terminus of said polypeptide and is atripeptide with the amino acid sequence Xaa-Pro-Pro, wherein Xaa is anyamino acid.
 39. The purified polypeptide of claim 38, wherein Xaa isAla.
 40. The purified polypeptide of claim 37, wherein said terminalstabilizing group is at the carboxy terminus of said polypeptide and isa tripeptide with the amino acid sequence Pro-Pro-Xaa, wherein Xaa isany amino acid.
 41. The purified polypeptide of claim 40, wherein Xaa isAla.
 42. The purified polypeptide of claim 37, wherein said terminalstabilizing group is a small stable protein.
 43. The purifiedpolypeptide of claim 42, wherein said small stable protein comprises afour-helix bundle topology.
 44. The purified polypeptide of claim 42,wherein said small stable protein is Rop.
 45. The purified polypeptideof claim 35, further comprising an Asp at the amino terminus of saidamino acid sequence.
 46. A composition comprising the polypeptide ofclaim
 35. 47. A purified polypeptide, the amino acid sequence of whichconsists of:(Xaa₁)_(n)-Xaa₂-Cys-Ala-Xaa₃-His-Xaa₄-Xaa₅-Xaa₆-Leu-Val-Trp-Cys-(Xaa₇)_(n),(SEQ ID NO:1), wherein Xaa₁ is absent or is any amino acid, Xaa₂ is Pheor Arg, Xaa₃ is any amino acid, Xaa₄ is Gly or Ala, Xaa₅ is Glu or Ala,and Xaa₆ is any non-aromatic amino acid.
 48. A purified polypeptide, theamino acid sequence of which consists of:(Xaa₁)_(n)-Cys-Ala-Xaa₂-His-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-(Xaa₃)_(n)(SEQ ID NO:35), wherein Xaa₁ is any amino acid, Xaa₂ is Arg, Trp, Tyr,or Phe, Xaa₃ is any amino acid, and n is 0, 1, 2, 3, 4, or
 5. 49. Apurified polypeptide, the amino acid sequence of which consists of:(Xaa₁)_(n)-Cys-Ala-Xaa₂-His-Xaa₃-Xaa₄-Xaa₅-Leu-Val-Trp-Cys-Xaa₆-(Xaa₇)_(n)(SEQ ID NO:34), wherein Xaa₁ is any amino acid, Xaa₂ is Phe or Arg, Xaa₃is any amino acid, Xaa₄ is Gly or Ala, Xaa₅ is Glu or Ala, Xaa₆ is anynon-aromatic amino acid, Xaa₇ is any amino acid, and n is 0, 1, 2, 3, 4,or 5.